Shc-binding protein

ABSTRACT

Novel Shc-binding protein, oligonucleotides encoding the same, methods of producing and use thereof are disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of U.S. application Ser. No. 09/083,587, filedMay 21, 1998.

FIELD OF INVENTION

The present invention relates generally to the identification andisolation of a novel Shc-binding proteins, to novel nucleic acidmolecules encoding such polypeptides and more particularly to theisolation and identification of a unique Shc binding protein designatedPAL Protein expressed in Activated Lymphocytes), and nucleic acidmolecules encoding PAL.

BACKGROUND

The ubiquitously expressed Shc adapter proteins play a role in couplinggrowth factor receptor activation to intracellular signaling pathways.The mammalian Shc gene encodes at least three overlapping proteins withmolecular weights of approximately 46 kDa, 52 kDa and 66 kDa (alsocalled the p46, p52, and p66 isoforms or proteins, Bonfini et al., TIBS21:257-261 (1996); Migliaccio et al., EMBO J. 16:706-716 (1997); Pelicciet al., Cell 70:93-104 (1992)). All three protein products share acarboxy terminal Src Homology 2 (SH2) domain, a central glycine/prolinerich domain with homology to alphal collagen (CH1), and an aminoterminal phosphotyrosine binding (PTB) domain which is different fromthe SH2 domain. The p52 and p46 isoforms differ only by 46 amino acidsat the extreme amino terminus and are generated by the use ofalternative translation initiation sites (Pelicci et al., Cell 70:93-104(1992)). The p66 isoform is produced via alternative splicing of the Shcgene and contains an amino terminal extension which encodes a secondcollagen homology region (CH2) in addition to the common PTB, CH1, andSH2 domains. Interestingly, it has recently been demonstrated that theexpression of p66 is more restricted, and that some of its biological,properties are distinct from those of the p52 and p46 Shc isoforms(Bonfini et al., TIBS 21:257-261 (1996); Migliaccio et al., EMBO J.16:706-716 (1997)).

Shc proteins are typically tyrosine phosphorylated following activationof receptor tyrosine kinases (van der Geer et al., Ann. Rev. Cell Biol.10:251-337 (1994)), such as the epidermal growth factor receptor (EGFR)(Pelicci et al., Cell 70:93-104 (1992)), the platelet-derived growthfactor receptor (PDGFR) (Yokote et al., J. Biol. Chem. 269:15337-15343(1994)), the nerve growth factor receptor (TrkA) (Obermeier et al., EMBOJ. 13:1585-1590 (1994)), the insulin receptor (Pronk et al., J. Biol.Chem. 268:5748-5753 (1993)), and erbB-2 (Segatta et al., Oncogene9:2105-2112 (1993)), as well as following activation of receptors thatlack intrinsic tyrosine kinase activity, such as the T-cell receptor(TCR) (Ravichandran et al., Science 262:902-905 (1993)), the B-cellreceptor (Saxton et al., J. Immunol. 153:623-636 (1994)), the receptorsfor the interleukins (Burns et al., J. Biol Chem. 268:17659-17661(1993); Cutler et al., J. Biol. Chem. 268:21463-21465 (1993);Ravichandran et al., J. Biol. Chem. 269:1599-1602 (1994)), and theerythropoietin receptor (Damen et al., Blood 82:2296-2303 (1993)).Additionally, tyrosine phosphorylation of Shc proteins has been detectedafter activation of G-protein coupled receptors (Cazaubon et al., J.Biol. Chem. 269:24805-24809 (1994); Chen et al., EMBO J. 15:1037-1044(1996);al., Ptazniket et al. J. Biol. Chem. 270:19969-19973 (1995);Touhara et al., Proc. Natl. Acad. Sci. USA. 92:9284-9287 (1995); vanBiesen et al., Nature 376:781-784 (1995)), ligation of integrins(Maniero et al., EMBO J. 14:4470-4481(1995); Wary et al., Cell87:733-743 (1996)), and in cells expressing activated Src, Fps, Sea orLck (Baldari et al., Oncogene 16:1141-1147 (1995); Crowe et al.,Oncogene 9:537-544 (1994); McGlade et al., Proc. Natl. Acad. Sci. USA89:8869-8873 (1992); Pelicci et al., Oncogene 11:899-907 (1995)), whichimplicates Shc proteins as important substrates of cytoplasmic tyrosinekinases.

Shc Protein Binding

Shc proteins are able to directly bind to tyrosine-phosphorylatedpeptides or proteins, including activated receptor tyrosine kinases,typically by virtue of their SH2 or PTB domains (Bonfini et al., TIBS21:257-261 (1996); Pawson, Nature 373:573-579 (1995)). The Shc SH2domain preferentially binds to tyrosine phosphorylated peptides in thesequence context pY-E/I -X-I/L/M (where X represents any amino acid)(Songyang et al., Mol. Cell. Biol. 14:2777-2785 (1994)) and mediates thebinding of Shc to the PDGFR (Songyang et al., Mol. Cell. Biol.14:2777-2785 (1994)), EGFR (Pelicci et al., Cell 70:93-104 (1992)),receptor tyrosine kinase (RET) (Pronk et al. J. Biol. Chem.268:5748-5753 (1993)), and the CD3 zeta chain (Ravichandran et al.,Science 262:902-905 (1993)). The PTB domain of Shc proteins alsorecognizes tyrosine phosphorylated peptides, but in a different contextby selecting specific residues amino terminal to the phosphorylatedtyrosine (Songyang et al., J. Biol. Chem. 270:14863-14866 (1995)). TheShc PTB domain has been shown to bind directly to N-P-X- pY sequencemotifs in the cytoplasmic domains of the EGFR (Blaikie et al., J. Biol.Chem. 269:23031-32034 (1994)), TrkA(Diklic et al., J. Biol. Chem.270:15125-15129(1995)), RET (Lorenzo et al., Oncogene 14:763-771(1997)),and the IL-2Rβ chain (Ravichandran et al., Proc. Natl. Acad Sci. USA28:5275-5280 (1996)).

Phosphorylated Shc proteins are also able to associate with the Grb2adapter protein by binding of the Grb2 SH2 domain to the phosphorylatedtyrosine residue 317 (Y317) within the CH1 domain of Shc proteins(Rozakis-Adcock et al., Nature 360:689-692 (1992); Salcini et al.,Oncogene 9:2827-2836 (1994)). Grb2 is stably associated with the Rasguanine nucleotide exchange factor, SOS (Batzer et al., Nature 363:85-88(1993); Buday et al., Cell 73:611-620 (1993); Chardin et al., Science260:1338-1343 (1993); Egan et al., Nature 363:45-51 (1993);Rozakis-Adcock et al., Nature 363:83-85 (1993)), and membranelocalization of the Grb2-SOS complex results in activation of Ras(Aronheim et al., Cell 78:949-961 (1994)). Therefore, it has beenproposed that Shc proteins are involved in coupling cell surfacereceptors to Ras activation. Several studies on the effects of Shcprotein over expression provide support for this hypothesis. First,co-expression of a dominant negative mutant of Ras blocks neuriteoutgrowth in PC12 cells induced by Shc protein over expression(Rozakis-Adcock et al., Nature 360:689-692 (1992)). Second,over-expression of Shc protein in NIH 3T3 fibroblasts results intransformation (Pelicci et al., Cell 70:93-104 (1992)), and this can beabrogated by mutation of the presumed Grb2 binding site (Salcini et a.,Oncogene 9:2827-2836 (1994)). Also, over-expression of Shc proteinenhances EGF induced activation of MAP kinases (Migliaccio et al., EMBOJ. 16:706-716 (1997)), and cell motility and growth in response tohepatocyte growth factor (HGF) (Pelicci et al., Oncogene 10:1631-1638(1995)).

Shc Protein Ras-independent Binding

The modular structure of Shc proteins permits their interaction withmultiple signaling molecules, suggesting that Shc proteins couldfunction to couple activated receptors to pathways other than Ras. Twoadditional sites of Shc protein tyrosine phosphorylation have recentlybeen mapped to tyrosine residues 239 and 240 (Y239/240) (Gotoh et al.,EMBO J. 15:6197-6204 (1996); Harmer et al., Mol. Cell. Biol.17:4087-4095 (1997); van der Geer et al. Curr. Biol. 6:1435-1444(1996)). Tyrosine 239 is present within a Grb-2 SH2 binding motif, andhas been demonstrated to associate with Grb-2 in vivo (Gotoh et al.,Mol. Cell. Biol. 17:1824-1831 (1997); Harmer et al. Mol. Cell. Biol.17:4087-4095 (1997)). The Y239/240 phosphorylation sites may also coupleShc proteins to additional downstream SH2 containing proteins, sincephosphopeptides corresponding to Y239/240 have been demonstrated to bindto a variety of as yet, unidentified proteins, in addition to Grb2 (vander Geer et al., Curr. Biol. 6:1435-1444 (1996)). A novel role for Shcproteins has been suggested in which phosphorylation of Y239/Y240 leadsto c-myc induction, and suppression of apoptosis in Ba/F3 cells, in aRas-independent manner (Gotoh et al., EMBO J. 15:6197-6204 (1996)).

The Shc PTB domain has been demonstrated to bind directly to thecytoplasmic enzyme SHIP, an SH2 domain containing inositol5-phosphatase, in response to growth factor and cytokine stimulation inhematopoietic cells (Damen et al., Proc. Natl. Acad. Sci. USA93:1689-1693 (1996); Kavanaugh et al., Curr. Biol. 6:438-445 (1996);Lioubin et al., Genes & Development 10: 1084-1095 (1996)). Furthermore,proline rich sequences in the Shc CH1 domain are proposed to mediate theinteraction between Shc proteins and the SH3 domain of eps8, a tyrosinephosphorylated protein involved in EGF receptor mediated signaling(Bonfini et al., TIBS 21:257-261 (1996); Matoskova et al., Mol. Cell.Biol. 15:3805-3812 (1995)). Therefore, it is likely that Shc proteinsparticipate in diverse signal transduction pathways by interacting withmultiple cytoplasmic signaling molecules. Shc proteins are generallybelieved to be involved in various cell proliferation pathways.Specifically, Shc proteins appear to be involved in signaling pathwaysthat lead to cell division.

SUMMARY OF THE INVENTION

The invention is directed to an isolated and purified Shc bindingprotein designated PAL (Protein expressed in Activated Lymphocytes) andto nucleic acids encoding the proteins.

In certain embodiments, the invention is directed to a PAL polypeptidecomprising the polypeptide set out as SEQ ID NO.:2; the polypeptide setout as SEQ ID NO. :4; a polypeptide that is at least 75 percentidentical to the foregoing polypeptides; and a biologically activefragment, homolog or variants, conserved variants, allelic variantanalogs. The PAL polypeptide optionally may have an amino terminalmethionine. The polypeptides of the invention may also be covalentlymodified with for example water soluble polymers, and fusion withpeptides preferably at the amino or carboxy terminus of a PALpolypeptide according to the invention. Also encompassed by theinvention are polypeptides having about 50% sequence similarity to thepolypeptides set out as SEQ ID NOS.:2 or 4.

The invention is further directed to anti-PAL antibodies directedagainst such PAL polypeptides.

In certain embodiments, the present invention is directed to apolynucleotide molecule encoding a polypeptide selected from the groupcomprising of the polynucleotide molecule of SEQ ID NO.:1; thepolynucleotide molecule of SEQ ID NO.:3; a polynucleotide moleculeencoding the polypeptide of SEQ ID NO.:2 or a biologically activefragment thereof; a polynucleotide molecule that encodes a polypeptidethat is at least 75 percent identical to the polypeptide of SEQ IDNO.:2; a polynucleotide molecule encoding the polypeptide of SEQ IDNO.:4 or a biologically active fragment thereof; a polynucleotidemolecule that encodes a polypeptide that is at least 75 percentidentical to the polypeptide of SEQ ID NO.:4; a polynucleotide moleculethat hybridizes under stringent conditions to a polynucleotide moleculecomplementary to any of items (a)-(f) below; and a polynucleotidemolecule that is the complement of any of items (a)-(g) below.

In other embodiments, the invention is directed to vectors comprisingthese polynucleotide molecules, and host cells, either prokaryotic oreukaryotic, comprising the vectors, and recombinant host cellscontaining the polynucleotide molecules or vectors according to theinvention.

In other embodiments, the invention is directed to a polynucleotide orfragment(s) thereof, which can be used to detect the presence ofpolynucleotide molecules that encode PAL polypeptides. In otherembodiments, the invention provides methods of using such nucleic acidsor fragments to detect the presence of nucleic acids encoding PALpolypeptides.

In other embodiments, the invention provides a process for producing aPAL polypeptide including the polypeptides described above.

In yet other embodiments, the invention is directed to a process forproducing a recombinant host cell that expresses a PAL polypeptideaccording to the invention, and to methods of producing PAL polypeptidesusing those host cells.

In still another embodiment, the invention is directed to a mammaliancell containing a PAL polypeptide encoding DNA and modified in vitro topermit higher expression of PAL polypeptide by means of a homologousrecombinational event consistent of inserting an expression regulatorysequence in functional proximity to the PAL encoding DNA therebyincreasing the expression of a PAL polypeptide by activating of anendogenous PAL gene.

In other embodiments, the invention provides a process of using suchrecombinant host cells to screen compounds or compositions for theirability to block cell division or proliferation.

In yet other embodiments, the invention provides a transgenic animalthat produces PAL polypeptide, according to the present invention on totransgenic animals in which one or more of its PAL genes is disrupted or“knocked out” and

The invention also provides a process of using such a transgenic animalto screen compounds or compositions for their ability to block celldivision or proliferation by measuring the effect on PAL production andinhibition of cell proliferation in vivo in the transgenic animal by thecompounds or compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the nucleic acid sequence of the cDNA encoding humanPAL (SEQ ID NO.:1).

FIGS. 2A-2F depict the deduced amino acid sequence for human PAL astranslated from the cDNA (SEQ ID NO.:2).

FIGS. 3A-3C depict the nucleic acid sequence of the cDNA encoding murinecDNA (SEQ ID NO.:3).

FIGS. 4A-4E depict the deduced amino acid sequence for murine PAL astranslated from the cDNA (SEQ ID NO.:4).

FIGS. 5A-5C depict the amino acid sequence comparison between human PALand the murine PAL polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

A novel Shc binding protein designated PAL (Protein expressed inActivated Lymphocytes) and nucleic acids encoding the protein aredisclosed herein. DNAs encoding mouse PAL (mPAL), and human PAL (hPAL)have been isolated and characterized. Such DNAs may be used to obtainPAL DNAs from other species using methods well known in the art. Theterm PAL is used generically and is not intended to be species specific.

The predicted amino acid sequence encoded by mPAL DNA contains 23tyrosine residues, several of which are embedded in consensus bindingmotifs for SH2 domains. In addition, two highly acidic regions areencoded by the mPAL DNA. Comparison of both the nucleotide and proteinsequences of mPAL with the GenBank databases revealed no significanthomology between mPAL and any previously identified proteins. We haveidentified several related expressed sequence tags (ESTs) representhuman and rat homologues of mPAL. In addition several short murine andhuman ESTs with approximately 50% sequence similarity to regions of mPALwere identified, suggesting that additional mPAL related genes exist.

PAL binds specifically to the Shc SH2 domain and, unlike previouslydescribed Shc SH2-protein interactions, the association of PAL and Shcprotein is phosphotyrosine independent. Both PAL RNA and proteinexpression are restricted to tissues containing actively dividing cellsand to proliferating cells in culture. PAL expression is induced upongrowth factor stimulation and is down regulated upon growth inhibition.This pattern and timing of PAL expression and its association with theShc protein, suggests a role for this protein in signaling pathwaysgoverning cell cycle progression.

PAL is also more highly expressed in tumor cell lines than in normallyproliferating cell lines, which indicates that PAL, or nucleic acidsthat encode all or a portion of PAL, may serve as tumor markers fordiagnosing cancer or localizing tumor cells. Moreover, the PAL may alsobe used as a target in test systems, such as transformed cell lines ortransgenic animals, to screen for drugs that may be useful for blockingthe promotion of cell proliferation, more particularly in screeningcandidate drugs for cancer treatment.

There are also circumstances where cell division or proliferation aredesirable, such as in the stimulation of hematopoiesis followingchemotherapy or radiation therapy or to encourage or stimulate growth ofcultured mammalian cells. In such settings, PAL may be usedtherapeutically to stimulate cell proliferation either by theintroduction of expressible PAL encoding DNA into cells (gene therapy)or by the use of PAL polypeptides to directly stimulate hematopoiesiseither in an in vivo or ex vivo context.

Yet a further aspect of the present invention is to provide nucleic acidmolecules encoding PAL polypeptides, and encompassed in the presentinvention are methods of preparing such nucleic acid molecules andpolypeptides.

The present invention also provides methods of detecting or determiningthe presence of tumor cells using nucleic acids that encode all or aportion of PAL polypeptides or using antibodies directed against PALpolypeptides.

Another aspect of the present invention provides methods of screeningcompounds or compositions for their ability to block cell division orproliferation using cell lines or transgenic animals that comprisenucleic acids that produce PAL polypeptides. Such cell lines and suchtransgenic animals are also provided according to certain embodiments ofthe present invention.

Also included in the scope of this invention are PAL polypeptides suchas the polypeptide of SEQ ID NO.:2 and SEQ ID NO.:4 and relatedbiologically active polypeptide fragments, homologs, variants, conservedvariants, allelic variants and derivatives thereof. Further includedwithin the scope of the present invention are nucleic acid moleculesthat encode these polypeptides, and methods for preparing thepolypeptides.

The invention is also directed to non-human mammals such as mice, rats,other rodents, rabbits, goats, sheep, and other animals including farmanimals, in which one or both of copies of the gene encoding theanimal's equivalent of human PAL has been disrupted (“knocked out”) soas to prevent expression of an active gene product or to significantlyreduce the activity of an expressed gene product. Such mammals may beprepared using techniques and methods such as those described in U.S.Pat. No. 5,557,032, incorporated herein by reference.

The present invention further includes non-human mammals such as mice,rats, other rodents, rabbits, goats, sheep, and other farm animals inwhich the gene (or genes) encoding PAL polypeptides (either the nativeform of PAL for the mammal or a heterologous PAL gene), is overexpressed by the mammal by way of the introduction of expressionregulatory sequences in functional proximity to the animal's endogenousPAL gene or by introducing into the animal a transgene comprising anexpression regulatory sequence and a PAL encoding DNA, thereby creatinga “transgenic” mammal. Such transgenic mammals may be prepared usingwell known methods such as those described in U.S. Pat. No. 5,489,743and PCT patent application No. WO94/28122, published Dec. 8, 1994.

The term “PAL protein” or “PAL polypeptide” as used herein refers to anyprotein or polypeptide having the properties described herein for PAL.The PAL polypeptide may or may not have an amino terminal methionine,which may depend on the manner in which it is prepared. By way ofillustration, PAL protein or PAL polypeptide refers to (1) an amino acidsequence encoded by PAL nucleic acid molecules as defined in any ofitems (a)-(f) below, and peptide or polypeptide fragments derivedtherefrom, (2) naturally occurring allelic variants of the PAL genewhich result in one or more amino acid substitutions, deletions, and/orinsertions as compared to the PAL polypeptide of SEQ ID NO.:3 or SEQ IDNO.:4, and/or (3) chemically modified derivatives as well as nucleicacid and or amino acid sequence variants thereof as provided for herein.

As used herein, the term “PAL fragment” refers to a peptide orpolypeptide that is less than the full length amino acid sequence ofnaturally occurring PAL protein but has a biological activity of orsimilar to PAL polypeptide or PAL protein described above. Such afragment may be truncated at the amino terminus, and/or the carboxyterminus, and/or internally, and may be chemically modified. Such PALfragments may or may not include an amino terminal methionine. PALfragments also include immunologically active fragments of the PALpolypeptide, the fragments capable of eliciting antibody response in ahost animal.

As used herein, the term “PAL derivative” or “PAL variant” refers to aPAL polypeptide, protein, or fragment that 1) has been chemicallymodified, as for example, by addition of one or more polyethylene glycolmolecules, sugars, phosphates, polypeptides (i.e. fusion proteinsincluding fusion with immuoglobulin molecules or fragments thereof suchas is described in WO97/24440, incorporated herein by reference,), orother such molecules not naturally attached to wild-type PALpolypeptide, the modifications may be covalent modifications, a “PALvariant” that 2) contains one or more nucleic acid or amino acidsequence substitutions, deletions, and/or insertions as compared to thePAL nucleic acid or amino acid sequence set forth in SEQ ID No.:1 or 2.The PAL polypeptide(s) or fragment(s), variant(s) including the variantsdiscussed above or homolog(s) of the PAL polypeptide(s) may bechemically modified, i.e., glycosylated, phosphorylated, and/or linkedto a polymer, as described below, they may have an amino terminalmethionine, depending on how they are prepared and may also comprise afusion protein or fusion polypeptide.

The full length PAL polypeptide or fragment thereof can be preparedusing well known recombinant DNA technology methods such as those setforth in Sambrook et al. Molecular Cloning. A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) and/orAusubel et al., eds, Current Protocols in Molecular Biology, GreenPublishers Inc. and Wiley and Sons, NY (1994). A gene or cDNA encodingthe PAL protein or fragment thereof may be obtained for example byscreening a genomic or cDNA library, or by PCR amplification.Alternatively, a gene encoding the PAL polypeptide or fragment may beprepared by chemical synthesis using methods well known to the skilledartisan such as those described by Engels et al., Angew. Chem. Intl.Ed., 28:716-734 (1989). These methods include, inter alia, thephosphotriester, phosphoramidite, and H-phosphorate methods for nucleicacid synthesis. A preferred method for such chemical synthesis ispolymer-supported synthesis using standard phosphoramidite chemistry.Typically, the DNA encoding the PAL polypeptide will be several hundrednucleotides in length. Nucleic acids larger than about 100 nucleotidescan be synthesized as several fragments using these methods. Thefragments can then be ligated together to form the full length PALpolypeptide. Usually, the DNA fragment encoding the amino terminus ofthe polypeptide will have an ATG, that encodes a methionine residue.This methionine may or may not be present on the mature form of the PALpolypeptide, depending on whether the polypeptide produced in the hostcell is secreted from that cell.

In some cases, it may be desirable to prepare nucleic acid and/or aminoacid variants or analogs of naturally occurring PAL. Nucleic acidvariants or analogs (wherein one or more nucleotides and/or amino acidsare designed to differ from the wild-type or naturally occurring PAL)may be produced using site directed mutagenesis or PCR amplificationwhere the primer(s) have the desired point mutations (see Sambrook etal., supra, and Ausubel et al., supra, for descriptions of mutagenesistechniques). Chemical synthesis using methods described by Engels etal., supra, may also be used to prepare such variants. Other methodsknown to the skilled artisan may be used as well. For example, in Wayneet al., EMBO J 2:1827-1829 (1983), the authors teach a method fordeletion mutagenesis, that was used to generate mutants of the TyrTSgene. Huang et al., Cell 48:129-136 (1987), analyzed the functionaldomains of Pseudomonas exotoxin using a deletion analysis of the geneexpressed in E. coli. In 1986, Zumstein et al., J. Mol. Biol. 191:333-340, described the analysis of structural and functional domains ofE. coli DNA Topoisomerase I using insertion and deletion mutagenesis. InDeChiara et al., Methods in Enzymol. 119:403-415, the authors describeprocedures for in vitro DNA mutagenesis of human leukocytes interferon.Other publications describing mutagenesis of cloned genes and subsequenttesting of the polypeptide encoded thereby include Doyle et al., J. CellBiol. 103:1193-1204 (1986), and others. Preferred nucleic acid variantsor analogs are those containing nucleotide substitutions accounting forcodon preference in the host cell that is to be used to produce PAL.Other preferred variants or analogs are those encoding conservativeamino acid changes as described above (e.g., wherein the charge orpolarity of the naturally occurring amino acid side chain is not alteredsubstantially by substitution with a different amino acid), as comparedto wild type, analogs of PAL polypeptide(s), and/or those designed toeither generate a novel glycosylation and/or phosphorylation site(s) onPAL, or those designed to delete an existing glycosylation and/orphosphorylation site(s) on PAL.

As used herein, the terms “biologically active” when used in the contextof any polypeptide(s), fragment(s), derivative(s), homolog(s) andvariant(s) refers to a molecule having a biological activity of PAL suchas the ability to stimulate cell growth or division if either in vivo orin vitro as well as affecting signaling pathways governing cell cycleprogression. According to the present invention the ability to induceantibody production in a suitable host is also within the meaningof“biological activity” and “immunologically active”. As used herein,the terms “effective amount” and “therapeutically effective amount”refer to the amount of PAL necessary to support one or more biologicalactivities of PAL as set forth above. The PAL polypeptides that have usein practicing the present invention may be naturally occurring filllength polypeptides, or truncated polypeptides or variant homologs oranalogs or derivatives or peptide fragments. Illustrative analogsinclude those in which one or more divergent amino acids between twospecies are substituted with the divergent amino acid from anotherspecies. Divergent amino acids may also be substituted with any otheramino acid whether it be a conservative or a non-conservative aminoacid. More particularly PAL analogs may comprise the amino acid sequenceset out as SEQ ID NOS.:2 or 4, wherein one or more amino acids selectedfrom the group consisting of amino acids 4, 5, 6, 7, 10, 13, 14, 17, 19,21, 22, 23, 25, 27, 29, 32, 33, 34, 38, 40, 42, 46, 47, 49, 50, 51, 54,55, 56, 57, 58, 59, 60, 66, 69, 71, 75, 99 103, 104, 116, 125, 130, 132,136, 137, 148, 149, 150, 151, 153, 154, 159, 160, 161, 162, 171, 172,182, 186, 187, 189, 191, 203, 241, 288, 292, 321, 324, 326, 327, 331,332, 340, 341, 348, 349, 352, 354, 356, 357, 358, 361, 364, 369, 372,377, 378, 384, 395, 429, 431, 439, 443, 450, 452, 477, 478, 484, 499,503, 532, 535, 553, 559, 560, 562, 563, 564, 565, 570, 571, 573, 574,575, 577, 580, 582, 583, 588, 589, 590, 592, 593, 594, 598, 599, 601,602, 604, 605, 608, 609, 615, 619, 621, 623, 627, 628, 645, 652, 653,655, and 672 is substituted with another amino acid

As used herein, the term “PAL” when used to describe a nucleic acidmolecule refers to a nucleic acid molecule or fragment thereof that (a)has the nucleotide sequence as set forth in SEQ ID NO.:1 or SEQ IDNO.:3; (b) has a nucleic acid sequence encoding a polypeptide that is atleast 75 percent identical, but may be greater than 75 percent, i.e., 85percent, 95 percent, or even greater than 95 percent identical, to thepolypeptide encoded by any of SEQ ID NOS: 2 or 4; (c) is a naturallyoccurring allelic variant of (a) or (b); (d) is a nucleic acid variantof (a)-(c) produced as provided for herein; (e) has a sequence that iscomplementary to (a)-(d); and/or (f) hybridizes to any of (a)-(e) underhigh stringency conditions. The term “high stringency conditions” refersto hybridization and washing under conditions that permit only bindingof a nucleic acid molecule such as an oligonucleotide or cDNA moleculeprobe to highly homologous sequences. Exemplary stringent hybridizationconditions are as follows: hybridization at 65° C. in 3×SSC, 20 mmNaPO₄, pH 6.8 followed by washing at 55° C.-65° C. and washing 0.015 MNaCl, 0.005 M NaCitrate, and 0.1 percent SDS. It is understood by thoseof skill in the art that variation in these conditions occurs based onthe length and GC nucleotide content of the sequences to be hybridized.Formulas standard in the art are available for determining exacthybridization conditions. See Sambrook et al., supra. For example,another stringent wash solution is 0.2×SSC and 0.1 percent SDS used at atemperature of between 50° C.-65° C. Where oligonucleotide probes areused to screen cDNA or genomic libraries, the following stringentwashing conditions may be used. One protocol uses 6×SSC with 0.05percent sodium pyrophosphate at a temperature of 35° C.-62° C.,depending on the length of the oligonucleotide probe. For example, 14base pair probes are washed at 35° C.-40° C., 17 base pair probes arewashed at 450° C.-50° C., 20 base pair probes are washed at 52° C.-57°C., and 23 base pair probes are washed at 57° C.-63° C. The temperaturecan be increased 2-3° C. where the background non-specific bindingappears high. A second protocol utilizes tetramethylammonium chloride(TMAC) for washing oligonucleotide probes. One stringent washingsolution is 3 M TMAC, 50 mm Tris-HCI, pH 8.0, and 0.2 percent SDS. Thewashing temperature using this solution is a function of the length ofthe probe. For example, a 17 base pair probe is washed at about 45-500°C. PAL encoding nucleic acids also includes nucleic acid sequences thatencode PAL polypeptide or a fragment thereof, by the way of degeneratecodons.

Percent sequence identity can be determined by standard methods that arecommonly used to compare the similarity in position of the amino acidsof two polypeptides. By way of example, using a computer program such asBLAST or FASTA, the two polypeptides for which the percent sequenceidentity is to be determined are aligned for optimal matching of theirrespective amino acids (the “matched span”, which can include the fulllength of one or both sequences, or a predetermined portion of one orboth sequences). Each computer program provides a “default” openingpenalty and a “default” gap penalty, and a scoring matrix such as PAM250. A standard scoring matrix (see Dayhoff et al., Atlas of ProteinSequence and Structure, vol. 5, supp.3 (1978)), can be used inconjunction with the computer program. The percent identity can then becalculated using an algorithm contained in a program such as FASTA as:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\begin{matrix}\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {within}\quad {the}} \right. \\{\left. {{matched}\quad {span}} \right\rbrack + \left\lbrack {{number}\quad {of}\quad {gaps}\quad {introduced}} \right.}\end{matrix} \\{{into}\quad {the}\quad {longer}\quad {sequence}\quad {in}\quad {order}\quad {to}}\end{matrix} \\\left. {{align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

Polypeptides that are at least 70 percent identical will typically haveone or more amino acid substitutions, deletions, and/or insertions ascompared with wild type PAL. Usually, the substitutions will beconservative so as to have little or no effect on the overall netcharge, polarity, or hydrophobicity of the protein but optionally mayincrease the activity of PAL. Exemplary conservative substitutions areset forth in Table I below.

TABLE I Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Other variants of the polypeptide may be prepared by aligning a humanPAL polypeptide sequence with a mouse PAL polypeptide sequence (or PALfrom other species) and identifying the divergent amino acids (FIG. 5).One or more of the divergent amino acids can then be substituted withthe diverging amino acid or with other amino acids. Such variants maytherefore be a composite polypeptide comprising amino acid sequencesderived PAL polypeptides derived from two or more species.

The PAL gene or cDNA can be inserted into an appropriate expressionvector for expression in a host cell. The vector is typically selectedto be functional in the particular host cell employed (i.e., the vectoris compatible with the host cell machinery such that amplification ofthe PAL gene and/or expression of the gene can occur). The PALpolypeptide or fragment thereof may be amplified/expressed inprokaryotic, yeast, insect (baculovirus systems), and/or eukaryotic hostcells. Selection of the host cell will depend at least in part onwhether the PAL polypeptide or fragment thereof is to be glyrosylated.If so, yeast, insect, or mammalian host cells are preferable; yeastcells will glycosylate the polypeptide, and insect and mammalian cellscan glycosylate and/or phosphorylate the polypeptide as it naturallyoccurs on the PAL polypeptide (i.e., “native” glycosylation and/orphosphorylation).

Typically, the vectors used in any of the host cells will comprise apromoter operatively linked usually to the 5′ end of a DNA molecule tobe expressed. Vectors also typically comprise other regulatory elementsas well such as an enhancer(s), an origin of replication element, atranscriptional termination element, a complete intron sequencecontaining a donor and acceptor splice site, a signal peptide sequence,a ribosome binding site element, a polyadenylation sequence, apolylinker region for inserting the nucleic acid encoding thepolypeptide to be expressed, and a selectable marker element. Each ofthese elements is discussed below. Optionally, the vector may contain a“tag” sequence, i.e., an oligonucleotide sequence located at the 5′ or3′ end of the PAL coding sequence that encodes poly-Histidine (such ashexahis), or another small sequences which may be immunogenic or whichmay have other biological properties such as the ability to prolong thehalf-life of the polypeptide or to target the polypeptide to cells,organelles or ligands. This tag will be expressed along with theprotein, and can serve as an affinity tag for purification of the PALpolypeptide from the host cell. Optionally, the tag can subsequently beremoved from the purified PAL polypeptide by various means such as usinga selected peptidase for example.

The 5′ flanking sequence may be homologous (i.e., from the same speciesand/or strain as the host cell), heterologous (i.e., from a speciesother than the host cell species or strain), hybrid (i.e., a combinationof 5′ flanking sequences from more than one source), synthetic, or itmay be the native PAL 5′ flanking sequence. As such, the source of the5′ flanking sequence may be any unicellular prokaryotic or eukaryoticorganism, any vertebrate or invertebrate organism, or any plant,provided that the 5′ flanking sequence is functional in, and can beactivated by the host cell machinery. The 5′ flanking sequence maycomprise of a tissue specific promoter which directs the expression ofthe encoded polypeptide in specific cells of tissues.

The 5′ flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically, 5′flanking sequences useful herein other than the PAL 5′ flanking sequencewill have been previously identified by mapping and/or by restrictionendonuclease digestion and can thus be isolated from the proper tissuesource using the appropriate restriction endonucleases. In some cases,the full nucleotide sequence of the 5′ flanking sequence may be known.Here, the 5′ flanking sequence may be synthesized using the methodsdescribed above for nucleic acid synthesis or cloning.

Where all or only a portion of the 5′ flanking sequence is known, it maybe obtained using PCR and/or by screening a genomic library withsuitable oligonucleotide and/or 5′ flanking sequence fragments from thesame or another species.

Where the 5′ flanking sequence is not known, a fragment of DNAcontaining a 5′ flanking sequence may be isolated from a larger piece ofDNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion using one or more carefully selected enzymes to isolate theproper DNA fragment. After digestion, the desired fragment may beisolated by agarose gel purification, Qiagen® column or other methodsknown to the skilled artisan. Selection of suitable enzymes toaccomplish this purpose are readily determined by one of ordinary skillin the art.

The origin of replication element is typically a part of prokaryoticexpression vectors purchased commercially that aids in the amplificationof the vector in a host cell. Amplification of the vector to a certaincopy number can, in some cases, be important for optimal expression ofthe PAL polypeptide. If the vector of choice does not contain an originof replication site, one may be chemically synthesized based on a knownsequence, and ligated into the vector.

The transcription termination element is typically located 3′ of the endof the PAL polypeptide coding sequence and serves to terminatetranscription of the PAL polypeptide. Usually, the transcriptiontermination element in prokaryotic cells is a G-C rich fragment followedby a poly T sequence. While the element is easily cloned from a libraryor even purchased commercially as part of a vector, it can also bereadily synthesized using methods for nucleic acid synthesis such asthose described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture mediumor it may encode a protein whose expression may be determined byphysical means such as by fluorescence or by color or by histochemicalmeans. Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Marker genes controlling expressionproduct are detectable by physical means, and comprise of genes encodingthe fluorescent green protein. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene.

The ribosome binding element, commonly called the Shine-Dalgarnosequence (prokaryotes), or the Kozak sequence (eukaryotes), is necessaryfor translation initiation of mRNA. The element is typically located 3′to the promoter and 5′ to the coding sequence of the PAL polypeptide tobe synthesized. The Shine-Dalgarno sequence is varied but is typically apoly-purine (i.e., having a high A-G content). Many Shine-Dalgarnosequences have been identified, each of which can be readily synthesizedusing methods set forth above and used in a prokaryotic vector.

In those cases where it is desirable for PAL to be secreted from thehost cell, a signal sequence may be used to direct the PAL polypeptideout of the host cell where it is synthesized. Typically, the signalsequence is positioned in the coding region of PAL nucleic acidsequence, or directly at the 5′ end of the PAL coding region. Manysignal sequences have been identified, and any of them that arefunctional in the selected host cell may be used in conjunction with thePAL gene. Therefore, the signal sequence may be homologous orheterologous to the PAL nucleotide sequence, and may be homologous orheterologous to the PAL polypeptide sequence. Additionally, the signalsequence may be chemically synthesized using methods set forth above. Inmost cases, secretion of the polypeptide from the host cell via thepresence of a signal peptide will result in the removal of the aminoterminal methionine from the polypeptide.

In many cases, transcription of the PAL polypeptide may be increased bythe presence of one or more introns on the vector; this is particularlytrue where PAL is produced in eukaryotic host cells, especiallymammalian host cells. The introns used may be naturally occurring withinthe PAL nucleic acid sequence, especially where the PAL sequence used isa full length genomic sequence or a fragment thereof. Where the intronis not naturally occurring within the PAL DNA sequence (as for mostcDNAs), the intron(s) may be obtained from another source. The positionof the intron with respect to the 5′ flanking sequence and the PALcoding sequence is important, as the intron must be transcribed to beeffective. As such, where the PAL nucleic acid sequence is a cDNAsequence, the preferred position for the intron is 3′ to thetranscription start site, and 5′ to the poly-A transcription terminationsequence. Preferably for PAL cDNAs, the intron will be located on oneside or the other (i.e., 5′ or 3′ ) of the PAL coding sequence such thatit does not interrupt the coding sequence. Any intron from any source,including any viral, prokaryotic and eukaryotic (plant or animal)organisms, may be used to practice this aspect of the invention,provided that it is compatible with the host cell(s) into which it isinserted. Also included herein are synthetic introns. Optionally, morethan one intron may be used in the vector.

Where one or more of the elements set forth above are not alreadypresent in the vector to be used, they may be individually obtained andligated into the vector. Methods used for obtaining each of the elementsare well known to the skilled artisan and are comparable to the methodsset forth above (i.e., synthesis of the DNA, library screening, and thelike).

The final vectors used to practice this invention are typicallyconstructed from a starting vectors such as a commercially availablevector. Such vectors may or may not contain some of the elements to beincluded in the completed vector. If none of the desired elements arepresent in the starting vector, each element may be individually ligatedinto the vector by cutting the vector with the appropriate restrictionendonuclease(s) such that the ends of the element to be ligated in andthe ends of the vector are compatible for ligation. In some cases, itmay be necessary to “blunt” the ends to be ligated together in order toobtain a satisfactory ligation. Blunting is accomplished by firstfilling in “sticky ends” using Klenow DNA polymerase or T4 DNApolymerase in the presence of all four nucleotides. This procedure iswell known in the art and is described for example in Sambrook et al.,supra.

Alternatively, two or more of the elements to be inserted into thevector may first be ligated together (if they are to be positionedadjacent to each other), and then ligated into the vector.

One other method for constructing the vector calls for conducting allligations of the various elements simultaneously in one reactionmixture. Here, many nonsense or nonfunctional vectors will be generateddue to improper ligation or insertion of the elements. However, thefunctional vector may be identified and selected by restrictionendonuclease digestion.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and/or mammalian host cells. Suchvectors include, inter alia, pCRII (Invitrogen, San Diego, Calif.),pBSII (Stratagene, LaJolla, Calif.), and pETL (BlueBacII; Invitrogen).

After the vector has been constructed and a PAL nucleic acid has beeninserted into the proper site of the vector, the completed vector may beinserted into a suitable host cell for amplification and/or PALpolypeptide expression.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell, or a vertebrate cell).The host cell, when cultured under appropriate conditions, cansynthesize PAL protein which can subsequently be collected from theculture medium (if the host cell secretes it into the medium), ordirectly from the host cell producing it (if it is not secreted). Aftercollection, the PAL protein can be purified using methods such asmolecular sieve chromatography, affinity chromatography, and the like.

Selection of the host cell will depend in part on whether the PALprotein is to be glycosylated or phosphorylated (in which caseeukaryotic host cells are preferred), and the manner in which the hostcell is able to “fold” the protein into tertiary structure (e.g., properorientation of disulfide bridges, etc.) such that biologically activeprotein is produced. However, where the host cell does not synthesizeproperly folded biologically active PAL, the PAL may be “folded” aftersynthesis using appropriate chemical conditions as discussed below. Itis also well known in the art that the host cell in which a PAL encodingDNA molecule is expressed will affect the glycosylation pattern of theexpressed protein with the result being that the protein expressed in ahost cell other than that in which it is normally expressed will have adifferent (e.g. non-native), glycosylation pattern. However, suchpolypeptides may still maintain full or partial biological activity.

Suitable cells or cell lines may be of mammalian origin, such as Chinesehamster ovary cells (CHO) or mouse 3T3 cells. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening, product production, and purification are knownin the art. Other suitable mammalian cell lines, are the monkey COS-1and COS-7 cell lines, and the CV-1 cell line. Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells may be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include but are notlimited to, human epitheloid carcinoma cell line, HeLa (ATCC CCL-2),mouse L-929 cells, 3T3 lines derived from Swiss (ATCC CRL-1658), Balb-cor NIH3T3 mice, BHK (ATCC CRL-10 or CRL-8544), or HaK (ATCC CRL-15),hamster cell lines all of which are available from the American TypeCulture Collection, in Rockville, Md.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, DH5α, DH10, and MC1061) are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Pseudomonas spp.,other Bacillus spp., Streptomyces spp., and the like may also beemployed in this method.

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect SF9 cells may beutilized as host cells in the method of the present invention (Miller etal., Genetic Engineering 8:277-298 (1986)).

Insertion (also referred to as “transformation” or “transfection”), ofthe vector into the selected host cell may be accomplished using suchmethods as calcium chloride, electroporation, microinjection,lipofection or the DEAE-dextran method. Alternatively, a desired genemay also be cloned into an appropriate “retroviral” or “adenoviral”vector. The desired gene can then be introduced into a host cell byinfection. The method selected will in part be a function of the type ofhost cell to be used and the result desired. These methods and othersuitable methods are well known to the skilled artisan, and are set fortfor example, in Sambrook et al., supra, and in Ausubel et al., supra. Inanother aspect of the invention, the host cells comprising a PAL genemay be used to insert in operative proximity to the endogenous gene,promoter regulatory sequences which increase the level of expression ofthe PAL gene. Typically, such promoter insertions are accomplished usinghomologous recombinations. See, for example, PCT InternationalPublication No. WO 94/12650, PCT International Publication No. WO92/20808, and PCT International Publication No. 91/09955.

The host cells containing the vector or the insertional promoterregulatory sequences (i.e., transformed or transfected), may be culturedusing standard media well known to the skilled artisan. The media willusually contain all nutrients necessary for the growth and survival ofthe cells. Suitable media for culturing E. coli cells are for example,Luria Broth (LB), and/or Terrific Broth (TB). Suitable media forculturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which may besupplemented with serum and/or growth factors as required by theparticular cell line being cultured. A suitable medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent on the plasmid with which the host cell was transformed. Forexample, where the selectable marker element is kanamycin resistance,the compound added to the culture medium will be kanamycin.

The amount of PAL polypeptide produced in the host cell can be evaluatedusing standard methods known in the art. Such methods include, withoutlimitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays such as DNA binding gelshift assays.

If the PAL polypeptide has been designed to be secreted from the hostcells, the majority of polypeptide may be found in the cell culturemedium. Polypeptides prepared in this way will typically not possess anamino terminal methionine, as it is removed during secretion from thecell. If however, the PAL polypeptide is not secreted from the hostcells, it will be present in the cytoplasm (for eukaryotic, grampositive bacteria, and insect host cells) or in the periplasm(for gramnegative bacteria host cells), and may have an amino terminalmethionine. Methods for designing vector constructs which result in arecombinant protein lacking a N-terminal methionine are known.

For intracellular PAL protein, the host cells are typically firstdisrupted mechanically or osmotically to release the cytoplasmiccontents into a buffered solution. PAL polypeptide can then be isolatedfrom this solution.

Purification of PAL polypeptide from solution can be accomplished usinga variety of techniques. If the polypeptide has been synthesized suchthat it contains a tag such as hexahistidine (PAL/hexaHis), or othersmall peptide at either its carboxyl or amino terminus, it may bepurified in a one-step process by passing the solution through anaffinity column where the column matrix has a high affinity for the tagor for the polypeptide directly (i.e., a monoclonal antibodyspecifically recognizing PAL). For example, polyHistidine binds withgreat affinity and specificity to nickel, thus an affinity column ofnickel (such as the Qiagen nickel columns) can be used for purificationof PAL/polyHis. (See for example, Ausubel et al., eds., CurrentProtocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, NewYork (1993)).

Where the PAL polypeptide has no tag, and where there are no anti-PALantibodies available, other well known procedures for purification canbe used. Such procedures include, without limitation, ion exchangechromatography, molecular sieve chromatography, HPLC, native gelelectrophoresis in combination with gel elution, and preparativeisoelectric focusing (“Isoprime” machine/technique, Hoefer Scientific).In some cases, two or more of any of the foregoing techniques may becombined to achieve increased purity. Preferred methods for purificationinclude polyHistidine tagging and ion exchange chromatography incombination with preparative isoelectric focusing.

If it is anticipated that the PAL polypeptide will be found primarily inthe periplasmic space of the bacteria or the cytoplasm of eukaryoticcells, the contents of the periplasm or cytoplasm, including inclusionbodies (erg., gram-negative bacteria), if the processed polypeptide hasformed such complexes, can be extracted from the host cell using anystandard technique known to the skilled artisan. For example, the hostcells can be lysed to release the contents of the periplasm by Frenchpress, homogenization, and/or sonication. The homogenate can then becentrifuged.

If the PAL polypeptide has formed inclusion bodies in the periplasm, theinclusion bodies can often bind to the inner and/or outer cellularmembranes and thus will be found primarily in the pellet material aftercentrifugation. The pellet material can then be treated with achaotropic agent such as guanidine or urea to release, break apart, andsolubilize the inclusion bodies. The PAL polypeptide in its now solubleform can then be analyzed using gel electrophoresis, immunoprecipitationor the like. If it is desired to isolate the PAL polypeptide, isolationmay be accomplished using standard methods such as those herein setforth below and in Marston et al., Methods in Enzymol., 182:264-275(1990).

If PAL polypeptide inclusion bodies are not formed to a significantdegree in the periplasm of the host cell, the PAL polypeptide will befound primarily in the supernatant after centrifugation of the cellhomogenate, and the PAL polypeptide can be isolated from the supernatantusing methods such as those set forth above and/or below.

In those situations where it is preferable to partially or completelyisolate the PAL polypeptide, purification can be accomplished usingstandard methods well known to the skilled artisan. Such methodsinclude, without limitation, separation by electrophoresis followed byelectroelution, various types of chromatography (immunoaffinity,molecular sieve, and/or ion exchange), and/or high pressure liquidchromatography. In some cases, it may be preferable to use more than oneof these methods for complete purification.

In addition to preparing and purifying PAL polypeptide using recombinantDNA techniques, the PAL polypeptides, fragments, and/or derivativesthereof may be prepared by chemical synthesis methods (such as solidphase peptide synthesis), using methods known in the art such as thoseset forth by Merrifield et al., J. Am. Chem. Soc., 85:2149 (1964);Houghten et al., Proc Natl Acad. Sci. USA, 82:5132 (1985); and Stewartand Young, Solid Phase Peptide Synthesis, Pierce Chem. Co., Rockford,Ill. (1984). Such polypeptides may be synthesized with or without amethionine on the amino terminus. Chemically synthesized PALpolypeptides or fragments may be oxidized using methods set forth inthese references to form disulfide bridges. The PAL polypeptides orfragments may be employed as biologically active or immunologicalsubstitutes for natural, purified PAL polypeptides in therapeutic andimmunological processes.

Chemically modified PAL compositions (i.e., “derivatives”), where thePAL polypeptide is linked to a polymer (“PAL-polymers”), are includedwithin the scope of the present invention. The polymer selected istypically water soluble so that the protein to which it is attached doesnot precipitate in an aqueous environment, such as a physiologicalenvironment. The polymer selected is usually modified to have a singlereactive group, such as an active ester for acylation or an aldehyde foralkylation, so that the degree of polymerization may be controlled asprovided for in the present methods. Included within the scope ofPAL-polymers is a mixture of polymers. Preferably, for therapeutic useof the end- product preparation, the polymer will be pharmaceuticallyacceptable.

The water soluble polymer or mixture thereof may be selected from thegroup consisting of; for example, polyethylene glycol (PEG),monomethoxy-polyethylene glycol, dextran, cellulose, or othercarbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol), andpolyvinyl alcohol.

For the acylation reactions, the polymer(s) selected should have asingle reactive ester group. For reductive alkylation, the polymer(s)selected should have a single reactive aldehyde group. The polymer maybe of any molecular weight, and may be branched or unbranched. Aparticularly preferred water-soluble polymer for use herein ispolyethylene glycol, abbreviated PEG. As used herein, polyethyleneglycol is meant to encompass any of the forms of PEG that have been usedto derivatize other proteins, such as mono-(C1-C10) alkoxy oraryloxy-polyethylene glycol.

Pegylation (i.e. modification by the addition of PEG or a PEGderivative), of PAL may be carried out by any of the pegylationreactions known in the art, as described for example in the followingreferences: Focus on Growth Factors 3:4-10 (1992); EP 0 154 316; and EP0 401 384. Preferably, the pegylation is carried out via an acylationreaction or an alkylation reaction with a reactive polyethylene glycolmolecule (or an analogous reactive water-soluble polymer), as describedbelow.

In general, chemical derivatization may be performed under any suitableconditions used to react a biologically active substance with anactivated polymer molecule. Methods for preparing pegylated-PAL willgenerally comprise the steps of (a) reacting an PAL polypeptide withpolyethylene glycol (such as a reactive ester or aldehyde derivative ofPEG), under conditions whereby PAL becomes attached to one or more PEGgroups, and (b) obtaining the reaction product(s). In general, theoptimal reaction conditions for the acylation reactions will bedetermined based on known parameters and the desired result. Forexample, the larger the ratio of PEG: protein, the greater thepercentage of poly-pegylated product.

Generally, conditions which may be alleviated or modulated byadministration of the present polymer/PAL include those described hereinfor PAL molecules in general. However, the polymer/PAL moleculesdisclosed herein may have additional activities, enhanced or reducedactivities, or other characteristics, as compared to the non-derivatizedmolecules.

PAL nucleic acid molecules, fragments, and/or derivatives that do notthemselves encode polypeptides that are active in activity assays may beuseful as hybridization probes in diagnostic assays to test, eitherqualitatively or quantitatively, for the presence of PAL DNA or RNA inmammalian tissue or bodily fluid samples or to produce immunologicallyactive fragments of the polypeptide.

Assays to Screen for Inhibitors or Activators of PAL

In some situations, it may be desirable to inhibit or significantlydecrease the level of PAL activity. For instance, inhibiting or reducingthe level of PAL activity may be useful in cancer or tumor therapy.Compounds that inhibit PAL activity could be administered either in anex vivo manner, or in an in vivo manner by subcutaneous or intravenous(i.v.) injection, or by oral delivery, implantation device, or the like.The assays described below exemplify methods useful for identifyingcompounds that inhibit PAL activity.

For ease of reading, the following definition is used herein fordescribing the assays:

“Test molecule(s)” refers to the molecule(s) that is under evaluation asan inhibitor of PAL, typically by virtue of its potential ability toblock the interaction of PAL with Shc proteins.

Several types of in vitro assays using purified PAL protein orpolypeptide may be conducted to identify those compounds that perturbPAL activity. Such a perturbation may be accomplished by compounds thatfor example inhibit the interaction of PAL with Shc proteins.

In one such assay, purified PAL protein or a fragment thereof (preparedfor example using methods described above), can be immobilized byattachment to the bottom of the wells of a microtiter plate.Radiolabeled Shc protein, as well as the test molecule(s) can then beadded either one at a time or simultaneously to the wells. Afterincubation, the wells can be washed and counted using a scintillationcounter for radioactivity to determine the degree of PAL/Shc proteinbinding in the presence of the test molecule. Typically, the moleculewill be tested over a range of concentrations, and a series of control“wells” lacking one or more elements of the test assays are used foraccuracy in evaluating the results. A variation of this assay involvesattaching the Shc protein to the wells, and adding radiolabeled PALalong with the test molecule to the wells. After incubation and washing,the wells can be counted for radioactivity. Test compounds to decreasethe binding of Shc protein to PAL represent one class of inhibitors ofPAL activity.

Several means are available to “detectably label” PAL. For example, PALprotein can be radiolabeled using ¹²⁵I. Alternatively, a fusion proteinof PAL may be used wherein the DNA encoding PAL is fused to the codingsequence of a peptide such as the c-myc epitope. PAL-myc fusion proteincan readily be detected with commercially available antibodies directedagainst myc. The PAL protein may also be modified by fusion with animmunoglobulin or fragment thereof (e.g. F_(c) fragment), which may bedetected for example by well known methods. Other markers or labelsinclude chromogenic or fluorogenic markers.

An alternative to microtiter plate type of binding assays comprisesimmobilizing either PAL or Shc protein on agarose beads, acrylic beadsor other types of such inert substrates. The inert substrate to whichthe PAL or Shc protein is attached placed in a solution containing thetest molecule along with the complementary component (either Shc proteinor PAL protein), which has been radiolabeled or fluorescently labeled;after incubation, the inert substrate can be collected bycentrifugation, and the amount of binding between PAL and Shc proteincan be readily assessed using the methods described above.Alternatively, the inert substrate complex can be immobilized in acolumn and the test molecule and complementary component passed over thecolumn. Formation of the PAL/Shc protein complex can then be assessedusing any of the techniques set forth above, i.e., radiolabeling,antibody binding, or the like.

Another type of in vitro assay that is useful for identifying a moleculeto inhibit PAL activity is the Biacore Assay System (Pharmacia,Piscataway, N.J.), using a surface plasmon resonance detector system andfollowing the manufacturer's protocol. This assay essentially involvescovalent binding of either PAL or Shc protein to a dextran-coated sensorchip which is located in a detector. The test molecule and thecomplementary component can then be injected into the chamber containingthe sensor chip either simultaneously or sequentially, and the amount ofbinding of PAL/Shc protein can be assessed based on the change inmolecular mass which is physically associated with the dextran-coatedside of the of the sensor chip; the change in molecular mass can bemeasured by the detector system.

In some cases, it may be desirable to evaluate two or more testmolecules together for use in decreasing or inhibiting PAL activity. Inthese cases, the assays set forth above can be readily modified byadding such additional test molecule(s) either simultaneously with, orsubsequently to, the first test molecule. The remainder of steps in theassay can be as set forth above.

Additional assays may be used to determine whether test molecules candisrupt PAL/Shc protein interaction in cell lines. For example, as notedabove, PAL is more highly expressed in tumor cell lines than in normallyproliferating cell lines. According to certain embodiments, one mayexpose a cell line that highly expresses PAL and expresses Shc proteinto test molecules to determine whether PAL/Shc protein binding isreduced, whether the production of PAL in the cell line is reduced,and/or proliferation of the cell line is reduced. One can compare theeffects of the test molecule by comparing these factors (effect onPAL/Shc protein binding, production of PAL, and/or proliferation of thecell line), in the test cell line (i.e. the one exposed to the testmolecule), to a control cell line that is not exposed to the testmolecule.

For determining the effect on PAL/Shc protein binding or PAL production,one can remove proteins from the cell or sample and use methods similarto those above that tag or isolate PAL/Shc protein complexes or PAL. Forexample, one could use immunoaffinity purification technique, which mayor may not include the use of fusion constructs for the PAL and Shc.

Similarly, one can use a transgenic animal that over expresses PAL andexpresses Shc protein to determine whether test molecules reduce PAL/Shcprotein binding, reduce production of PAL in the transgenic animal,and/or reduce tumor growth in the transgenic animal. Conversely, assaysmay also be used to screen for activators of PAL activity. Suchcompounds may be useful in promoting cell growth and division both invitro and in vivo. A person of ordinary skill in the art would readilyrecognize that several of the foregoing assays which measure theinhibition of PAL may be readily adapted to measure increases in PALactivity. Activators of PAL gene transcription may readily determinedusing techniques such as reverse transcriptase-polymerase chain reactiontechniques, RNAse protection assays and the like. Increased levels ofPAL protein are also readily determined by well known techniques such asimmuno-affinity techniques.

Also comprehended by the present invention is the use of PAL gene and/orprotein(s) including conserved variants, allelic variants, and analogsin a method of stimulating hematopoietic progenitor cell proliferationeither in vivo by direct administration to the patient or ex vivo byfirst obtaining hematopoietic progenitor cells and treating them inculture before administration to a patient. Such treatment may includecombinations of PAL polypeptides with other hematopoietic growth factorsincluding but not limited to stem cell factor (SCF), G-CSF, GM-CSF, EPO,CSF-1, IL-1, IL2. IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IGF-1, leukemia inhibitory factor (LIF), as well as othercytokines. Such a use is envisioned in the treatment of a group ofhematopoietic stem cell disorders which are characterized by a reductionin functional marrow mass, including stromal and or hematopoieticprecursor cells.

The following examples are intended for illustration purposes only, andshould not be construed as limiting the scope of the invention in anyway.

EXAMPLES

Standard methods for library preparation, DNA cloning, and proteinexpression are set forth in Sambrook et al., eds. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), and in Ausubel et al., eds., Current Protocols inMolecular Biology, Wiley, New York, N.Y. (1995). Standard methods forcell culture are set forth in Jacoby et al., Methods in Enzymology: CellCulture, Academic Press Inc., San Diego, Calif. (1979).

In order to identify binding partners for p52 Shc protein a yeast-twohybrid system was used. When full length p52 Shc protein was used as a“bait”, a novel protein designated mPAL was isolated multiple times fromboth T-cell (3 clones), and 11.5 day mouse embryo libraries (8 clones).Further analysis of the interaction in the two-hybrid systemdemonstrated that mPAL interacts specifically with Shc. Furthermore,mPAL was shown to interact with the isolated SH2 region of p52 Shc butnot with its CH1 or PTB domains. One of the cDNAs isolated from the twohybrid screen, encoding nucleotides 42 through 2130 of mPAL, was used toscreen a mouse spleen library (Stratagene, LaJolla, Calif.), in anattempt to obtain 5′ and/or 3′ cDNA sequences. As this approach failedto yield any additional 5′ or 3′ cDNA sequences, 5′ and 3′ RACE (Frohmanet al., Proc. Natl. Acad Sci. USA. 85: 8998-9002 (1980)) were used toobtain additional mPAL nucleotide sequence.

The combined cDNA clones (designated mPAL ), encompass 2246 bp (SEQ IDNO.:3). The 2007 bp open reading frame of mPAL includes nucleotides 12through 2018. The first in frame methionine (at nucleotide 12), is agood match for the Kozak consensus sequence (Kozak et al., Annu. Rev.Cell. Biol. 8:197-225 (1992)), and is used here to designate theinitiation codon. Thus the cDNA of mPAL encodes a polypeptide of 668amino acids with a predicted molecular weight of 75,917 Daltons. Asecond in-frame methionine occurs 16 amino acids downstream from thefirst, and also appears to be a good match for the Kozak consensussequence. The use of this methionine codon as an initiation codonresults in a protein of 653 amino acids with a predicted molecularweight of 74,407 Daltons.

Example I Yeast-two Hybrid Screen

In order to identify binding partners for p52 Shc, a yeast two hybridassay system was used. The yeast two-hybrid assay is based on the factthat many eukaryotic transcriptional activators are composed of twophysically separable, functionally independent domains. The yeast GAL4transcriptional activator protein, for example, contains a DNA-bindingdomain (GAL4-DB), and a transcriptional activator domain (GAL4-TA). TheGAL4-DB recognizes and binds to a sequence (UAS), in the upstreamregions of GAL4-responsive genes, while the GAL4-TA interacts with othercomponents of the transcription machinery needed to initiatetranscription. Both domains are required to activate a gene and,normally, the two domains are part of the same protein. However, if thetwo domains are physically separated (e.g. by way of recombinant DNAtechnology), and expressed in the same host cell, the GAL4-DB and TApeptides do not directly interact with each other and cannot activateresponsive genes. (Ma et al. Cell 51:443-446 (1988)).

In a yeast two hybrid system, two different cloning vectors are used togenerate separate fusions of these GALA domains that potentiallyinteract with each other. The recombinant hybrid proteins (hybrid ofGAL4 domain and a potential binding protein,) are co-expressed in yeastand are targeted to the yeast nucleus. If the non-GAL4-portions of thetwo types of hybrid interact with each other, the GAL4-DB will betethered to GAL4-TA. As a result of this interaction, GAL4transcriptional activator will be functionally reconstituted and willactivate transcription of reporter genes having upstream GAL4 bindingsites making protein-protein interaction phenotypically detectable. Theyeast two-hybrid system has been used either to screen libraries for agene(s) encoding a novel protein(s) that interacts with a known targetprotein or to test two known, previously cloned proteins forinteraction. (Chien et al., Proc. Natl. Acad. Sci. 88:9578-9583 (1991),incorporated herein by reference).

To use the yeast two-hybrid system to isolate and identify novelShc-binding proteins, a full length human p52 Shc DNA (Pellici et al.,Cell 70:93-104 (1992)), was cloned into the pAS-1 vector to generate afusion between the target protein, Shc, and the DNA binding domain ofGAL4. This created the “bait”, GAL4-Shc hybrid fusion protein. The yeaststrain, (Y153), (Bai and Elledge, Methods in Enzymol. 273:331-347(1996)) was used for transformation and contained both HIS3 and lacZreporter genes driven by promoters containing GAL4 binding sites, andwas deleted for endogenous GAL4 (Bai et al., Methods in Enzymol.273:331-347 (1996) ). Yeast clones, transformed with pAS-1 GAL4-Shc,were screened for expression of the GAL4-Shc fusion protein by Westernblot analysis of yeast lysates using either monoclonal or polyclonalanti-Shc antibodies as discussed below in Example VI. The GAL4-Shcexpressing clones were assayed for transcriptional activation of HIS3gene based on their ability to grow on His- media, and were assayed fortranscriptional activation of lacZ, by measuring β-gal activity using acolorimetric assay. The yeast expressing GAL4-Shc alone were negativefor activation of GAL4 driven promoters by both criteria. In an attemptto screen for molecules that bind to Shc-protein “bait”, a plasmid cDNAlibrary from resting murine T-cells was obtained (Staudinger et al. J.Biol. Chem. 268: 4608-4611 (1993)). In this library, total cDNA obtainedfrom resting T-cells was fused to the transcription activation domain ofGAL4, GAL4-TA. Also, a mouse embryo GAL4-TA fusion library (Clontech,Palo Alto, Calif.), was used to transform yeast carrying GAL4-Shc DBfusion constructs. The cDNA libraries were transfected into yeastcarrying the Shc-GAL4 DB fusion constructs and clones were selected onHis⁻ media supplemented with 20 mM aminotriazole. After 72 hours,nitrocellulose replicas of the transfected colonies were made andassayed for β-galactosidase activity directly by a method well known inthe art. Sambrook et al., Supra From the 6×10⁶ clones screened from theT-cell library, and 3×10⁶ clones screened from the mouse embryo library,forty two positive clones were picked and the cDNAs isolated. Theisolated cDNAs were checked for insert size and introduced into a secondstrain of yeast (Y187)Bai and Elledge, Methods in Enzymol 273:331-347(1996)). The purpose of this step is twofold, first the isolated cDNAcan be tested for β-gal activity alone, or when mated with yeastcarrying either GAL-Shc or GAL4 can be fused to other unrelatedproteins. This eliminates any false positives. Matings between Y153carrying the GAL4-Shc plasmid and Y187 carrying the GAL4-cDNA fusionwere assayed for β-gal activity to confirm positive clones. Followingthis procedure, twenty one clones were eliminated as false positives.Clones were classified as false positives if they were positive forβ-gal activity on their own or when mated with Y153 carrying the DNAbinding domain alone.

The remaining twenty one clones isolated in this first round ofscreening, were classified as specifically interacting with Shc based onthe following criteria: 1) yeast expressing both the cDNA and Shc hybridproteins were able to grow on His-media and were positive for β-galactivity; 2) the isolated cDNA transformed in Y187 was negative foractivation of HIS3 and lacZ when mated to yeast carrying the GAL4 DNAbinding domain alone; and 3) when mated with Y153, containing GAL4-Shc,the ability to transactivate both reporter constructs restored, butmating of the cDNA constructs with other GAL4 fusions did not result inactivation of transcription.

Screening of a random primed library derived from mouse embryo (day11.5) (commercially available from Clontech), was also carried out. Useof a random primed library allows the detection of Shc binding proteinswhich require the amino terminal sequences for binding. Also, by using alibrary from a different tissue, cDNAs not represented in the T-celllibrary were detected. Finally, the repeat isolation of relatedmolecules from two different libraries support the legitimacy of theinteraction being detected.

One of the clones isolated when full length p52 Shc was used as a baitencoded a novel protein sequence designated mPAL. This clone wasisolated multiple times from both T-cell (3 clones) and 11.5 day mouselibraries (8 clones). Further analysis of the interaction in thetwo-hybrid system demonstrated that this molecule interacts specificallywith Shc and not with other non-specific GAL4 fusion proteins.

Example II cDNA Library Screening, Isolation of mPAL cDNA andIdentification of a Protein Product Encoded by mPAL cDNA

A mouse spleen cDNA library (Stratagene, LaJolla, Calif.), was screenedwith the mPAL cDNA isolated from the yeast-two hybrid screen describedabove. The mouse spleen library was plated as per manufacturer'sinstructions. Bacteriophage plaques were immobilized on nylon filters asdescribed in Sambrook et al., supra. Filters were initiallyprehybridized for a minimum of 4 hours at 42° C. in a solution of 50%formamide, 4×SSPE, 1% SDS, 0.5% skim milk powder, 10% dextran sulphateand 10 mg/ml sheared salmon sperm DNA. A cDNA probe consisting of 1522bp AfiIII/PstI fragment of the mPAL cDNA isolated from yeast two-hybridscreen was [³² P]-radiolabeled by random hexamer priming (Pharmacia).[³²P]-Radiolabeled probe was added at 10⁶ cpm/ml and the filters furtherincubated for 16 hours at 42° C. Blots were washed twice for 10 minutesat room temperature in 2×SSC, 0. 1%SDS, then twice at 56° C. in 0.1×SSC,0.1% SDS, and then exposed to film. Single phage plaques containinghybridizing cDNAs were isolated, re-plated and rescreened untilhomogenous populations of phage were obtained. The cDNAs were excisedfrom Lambda Zap II Vector as per manufacturer's instructions(Stratagene), and sequenced.

cDNAs corresponding to nucleotides 42 to 2130 encoding mPAL amino acids16-668, were subcloned into Bluescript SK⁻ (Stratagene), and utilizedfor in vitro transcription and translation described in more detailbelow. The protein product produced is called mPAL met2, and details forits production are provided below.

The remaining 5′ and 3′ cDNA ends of mPAL were identified using 5′ and3′ RACE (Frohman, M. A., RACE: Rapid Amplification of cDNA Ends, PCRProtocols: A Guide to Methods and Applications eds. Innis, N. A. et al.Academic Press, Inc. (1990)), of Marathon Ready cDNAs from murine embryoand spleen using the manufacturer's instructions (Clontech, Palo Alto,Calif.). A full length construct encoding mPAL amino acids 1-668 wascloned by PCR from murine embryo Marathon Ready cDNA and cloned directlyinto pCR2.1 (Invitrogen, LaJolla, Calif.). This construct was sequencedfor accuracy and was used for in vitro transcription and translationdescribed in more detail below. The protein product is called mPAL met1and details of its production are provided below.

Example III

Preparation of GST-Fusion Proteins

This example discusses the preparation and purification of certainfusion proteins that are used in other examples in this application. TheShc SH12 mutant (R397A) was generated by using standard PCR-basedsite-directed mutagenesis technique converting amino acid Arg397 toAla397. Higuchi, R., Recombinant PCR in PCR Protocols: A Guide toMethods and Applications, eds. Innis, M. A. et al. pp 177-183, AcademicPress (1990)). The mutagenesis primers used were:

5′ mutagenic primer: GAG TTC TTG GTG GCA GAG AGC ACG (SEQ ID NO.:11)

3′ mutagenic primer: CGT GCT CTC TGC CAC CAA GAA GTC (SEQ ID NO.:12)

The PCR product containing the R397A mutation was cloned using BamHI andEcoRI restriction sites into the GST fusion vector, pGEX-2T (Pharmacia).The PAL cDNA encoding amino acids 11 to 648 was cloned using XhoIrestriction sites into the pGEX4T3 vector (Pharmacia). AdditionalGST-fusion proteins; Shc PTB (Blaikie et al., J. Biol. Chem.269:23031-32034 (1994)); Shc SH2 (Pelicci et al., Cell 70:93-104(1992)); Grb2 SH2 (Rozakis-Adcock et al., Nature 360:689-692 (1992));Vav SH2 (Margolis et al., Nature 356:71-74 (1992)); GAP-N SH2, PLCγ-NSH2, PLCγ-C SH2 (Anderson et al., Science 250:979-982 (1990)); and p85-NSH2, p85-C SH2 (McGlade et al., Mol. Cell Biol. 12:991-997 (1992)) havebeen previously described. Nck SH2 contains amino acids 281-377 of humanNck (Lehmann et al., Nucl. Acids. Res. 18:1048 (1990)), and the GSTShc-CH1 contains amino acids 212-376 of human Shc.

GST-SH2 fusion proteins were prepared as follows: DH5 α strain ofEscherichia coli (E. coli), a strain commonly used in the art forplasmid transformation was used for the preparation of GST-SH2 fusionproteins. Transformation competent DH5α cells were prepared as describedin Sambrook et al. supra. Competent cells were transformed with aplasmid, pGEX-2T, carrying the GST-SH2 fusion gene. Followingtransformation, the cells were plated on an agar medium containingampicillin to select for transformants carrying the GST-SH2 fusion gene.Log-phase DH5α cells carrying the GST-SH2 fusion gene were grown in thepresence of 100 μg/ml ampicillin, and induced with 1 mMisopropylthiogalactopyranoside (IPTG), for 3 hours at 370° C. The cellswere pelleted by centrifugation, lysed in 1 ml of ice-cold NP-40 lysisbuffer (50 mM Hepes, pH 7.25, 150 mM NaCl, 2 mM EDTA, 100 μM ZnCl₂, 1%(v/v) Nonidet-P40, (NP40), 100 μM sodium pervanadate, 10% (v/v)glycerol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM Pefa-Bloc(Boehringer-Mannheim, Indianapolis, Ind.), sonicated on ice three timesfor 30 seconds, and the lysates clarified by centrifugation for at11,000 rpm, 4° C. The lysates were incubated with glutathione-Sepharosebeads (Pharmacia) for 30 minutes at 4° C., the beads were washed severaltimes with NP-40 lysis buffer, and were resuspended in an equal volumeof phosphate-buffered saline (PBS) containing 1 mM dithiothreitol (DTT).Each of the above mentioned GST-fusion proteins were isolatedessentially in the same manner as described. The purified GST-fusionproteins were qualitatively and quantitatively analyzed by SDS-PAGEfollowed by Coomassie staining, and comparison with known BSA standards.

Example IV Transfections

Human p52 Shc in plasmid vector pECE has been previously described(Pelicci et al., Cell 70:93-104 (1992)). The mouse p66 Shc cDNA wassubcloned into pcDNA1 (Invitrogen). The mPAL cDNA encoding amino acids11 to 648 was excised from mPAL by digestion with XhoI and subclonedinto pcDNA3.1 (Invitrogen). 293T cells in 10 cm culture dishes weretransfected with 10 μg of pECE-Shc and/or pcDNA3.1-mPAL expressionvectors using Lipofectin (Gibco-BRL) according to the manufacturer'sinstructions. Cells expressing the transfected Shc and/or mPAL geneswere selected and used to assay in vivo association between mPAL and Shcdescribed below (Example X).

Example V Antibodies

Anti-mPAL antibodies were raised in rabbits against the peptide sequenceMVPPRPDLAAEKEP (SEQ ID NO.:5), corresponding to amino acids 16 through29 in the deduced protein sequence of mPAL. An amino terminal cysteinewas added to this peptide sequence to facilitate conjugation to keyholelimpet haemocyanin (KLH). The KLH-mPAL conjugate was used to immunizerabbits. Harlow et al., in Antibodies: A Laboratory Manual, Cold SpringHarbor, U.S.A. (1988).

The same mPAL peptide sequence used for immunization was also coupled toa “Sulfolink” column (Pierce, Rockford, Ill.) through an added aminoterminal cysteine, and the column was used for affinity purification ofanti-mPAL antibodies according to the protocol supplied by themanufacturer (Pierce). Briefly, 3 ml of whole serum collected fromimmunized animals was incubated on a “Sulfolink” column at roomtemperature for 1 hour. Unbound proteins are eluted from the column with3×5 ml of phosphate buffered saline (PBS). Antibodies specifically boundto the column are eluted using an elution buffer (200 mM glycine, pH2.5). Antibody elution off the column is monitored using aspectrophotometer at A₂₈₀. The antibodies are dialyzed overnight againstPBS and stabilized by the addition of 0.1 mg/ml bovine serum albumin.The purified antibody was used for Western blotting andimmunoprecipitation.

Unless otherwise indicated, in examples in this application, crudeanti-mPAL antibodies was used at a concentration of 1:500 for Westernblot analysis and 20 μl per immunoprecipitation, and affinity purifiedantibody was used at a final concentration of 1 μg/ml for Westernblotting and at 2 μg per immunoprecipitation. Affinity purified anti-Shcantibodies (Pelicci et al., Cell 70:93-104 (1992)), were used at a 1:500dilution for Western blot analysis and 2 μg per immunoprecipitation. Insome experiments, a monoclonal anti-Shc antibody (TransductionLaboratories, Lexington, Ky.), was used at a dilution of 1:250. Theanti-phosphotyrosine antibody RC20H (Transduction Laboratories), wasused at a dilution of 1:2500 for Western blot analysis. Theantiphosphotyrosine antibody 4G10 (UBI) was used for Western blotanalysis at a dilution of 1:1000, anti-EGFR antibody (UpstateBiotechnology, Lake Placid, N.Y.), was used at a dilution of 1:500 forWestern blot analysis, and anti-β-tubulin antibody (Amersham, Arlingtonheights, Ill.), was used at a concentration of 1 μg/ml for Western blotanalysis.

Example VI Immunoprecipitation and Western Blot Analysis

Studies were undertaken to examine the expression of PAL protein in avariety of cell types. Unless otherwise indicated, the followingconditions were used for PAL immunoprecipitation and Western blotanalysis. For NIH/3T3, P19, and 293 cells a single 10 cm plate of nearconfluent cells was used for immunoprecipitation and Western blotanalysis. Each 10 cm plate of adherent cells was lysed in 1 ml of NP-40lysis buffer for 15 minutes at 4° C. Tissue lysates were alsohomogenized in NP-40 lysis buffer using a polytron homogenizer(Kinematica AG, Littau, Switzerland). Lysates were transferred to a 1.5ml microfuge tube and centrifuged at 10,000×g (maximum speed), for 10minutes at 4° C. Once clarified by microcentrifugation, the totalprotein in the lysate was quantified by standard procedures. Sambrook etal., supra; Ausubel et al., supra. Typically, a volume of lysatecontaining 1 mg of total protein was incubated with 20 μl crudeanti-mPAL antiserum or 2 μg affinity purified anti-mPAL antiserum and100 μl of 10% Protein A Sepharose (Sigma Chemical Co., St. Louis, Mo.),and the volume increased to 1 ml with NP-40 lysis buffer. Samples wereincubated at 4° C. with gentle rotation for 1 hour. Immune complexeswere then washed three times in 1 ml NP-40 lysis buffer. Samples wereboiled for 5 minutes in 40 μl reducing SDS-Laemmli sample buffer priorto loading onto a 10% polyacrylamide gel and separation by SDS-PAGE.Proteins were electrophoretically transferred to Immobilon-P (PVDF)membrane (Millipore, Bedford, Mass.), and incubated in a blockingsolution of 5% skim milk powder in TBST (20 mM Tris-HCl, pH 8.3, 150 mMNaCl, 0.05% Tween 20), for a minimum of 1 hour prior to addition ofantibody overnight at 4° C. Membranes were washed three times in TBSTand incubated for 1 hour at 4° C. with a 1:3000 dilution of HRP-coupledprotein A (Biorad, Hercules, Calif.). Following incubation withsecondary antibodies, membranes were washed three times in TBST anddeveloped using ECL (Amersham), using the manufacturer's instructions.

Anti-mPAL antibodies specifically recognized in vitro transcribed andtranslated mPAL (mPAL met2), both by immunoprecipitation and Westernblot analysis.

Anti-mPAL antibodies were also used for immunoprecipitations from themurine embryonic cell line, P19. Western blot analysis detected animmunoprecipitating protein of 75 kDa which migrated with approximatelythe same mobility as in vitro transcribed and translated mPAL.Immunoprecipitation of in vitro transcribed and translated mPAL as wellas mPAL immunoprecipitated from P19 cells was blocked by preincubationof the antibody with 50 μg of immunizing peptide, further demonstratingthe specificity of this antiserum.

The 75 kDa band detected by anti-mPAL in P 19 immunoprecipitates wasresolved into two closely migrating bands by SDS-PAGE. In order toinvestigate whether the doublet was generated by the use of twoinitiation codons, the migration of mPAL from P19 lysates was comparedto in vitro transcribed and translated proteins from two mPAL cDNAconstructs. T7 TNT-coupled reticulocyte lysate systems (Promega,Madison, Wis.), was used to transcribe and translate cDNA constructsencoding full length mPAL amino acids 1-668 (met 1) in the vector PCR2.1(Invitrogen), or amino acids 16-668 (met2), in the vector Bluescript SK⁻(Stratagene), according to the manufacturer's instructions. TNT mPALmet1 represents the predicted full length mPAL open reading frame andincludes the initiating methionines encoded at position 12 and 56 of themPAL cDNA sequence. TNT mPAL met2 lacks the methionine at position 12but contains the internal ATG at nucleotide position 56. The doubletobserved in P19 immunoprecipitates appears to correspond to two distinctimmunoreactive proteins produced from TNT mPAL met 1. The TNT mPAL met2produced only one band comigrating with the lower band in P19 and TNTmet1 immunoprecipitates. This suggests that methionines at positions 1and 16 may be used both in vitro and in vivo.

However, PAL was not immunoprecipitated from cell lines derived fromspecies other than mouse, suggesting that anti-mPAL antibodies arespecies specific. In fact, cloning of human PAL (as described below),has confirmed that the amino terminal sequence against which ouranti-mPAL antibodies were raised is not well conserved in the humanprotein sequence.

Example VII GST-Precipitation Experiments

This example describes conditions used for GST-precipitation experimentsthat are discussed in more detail below. Cell lysates were prepared from293T cells transiently-transfected with p52 Shc in pECE, p66 Shc inpcDNA1 and/or mPAL in pcDNA3.1 as described above (Example III).

Cell lysates were incubated with approximately 5 μg of GST-fusionprotein coupled to glutathione-Sepharose 4B beads for 2 hours at 4° C.The beads were washed 3 times with NP-40 lysis buffer, resuspended in 25μL of SDS-sample buffer, the proteins resolved on a 10%SDS-polyacrylamide gel, and transferred to Immobilon-P membrane(Millipore). Western blot analysis for the detection of Shc proteins wascarried out using affinity purified anti-Shc antibody at a 1:500dilution (usually 0.5-1 μg antibody/mL of blocking solution). SimilarlyWestern blot analysis for mPAL was carried out as described above (seeExample VI), using the immunoaffinity purified anti mPAL antibody at a1:500 dilution (usually 0.5-1 μg/mL blocking solution), detection ofimmunoreactive bands was carried out as described above. The GST fusionproteins were visualized with Coomasie blue staining.

Competition studies assessing the phosphotyrosine dependence of GST-ShcSH2 and mPAL association were carried out in the presence of 10, 50 and100 mM O-phospho-L-threonine, O-phospho-L-serine, orO-phospho-L-tyrosine which were added to the cell lysates prior toincubation with immobilized GST-Shc SH2 fusion protein. WhileO-phospho-L-tyrosine inhibited the association of GST-Shc SH2 and mPAL,O-phospho-L-threonine and O-phospho-L-serine had no effect on theassociation of GST-Shc SH2 and mPAL.

Example VIII Phosphatase Treatment of PAL-Shc Complexes

PAL was precipitated from mPAL transfected 293T cells with 5 μg ofimmobilized GST-Shc SH2, washed twice with NP-40 lysis buffer, twicewith potato acid phosphatase (PAP) buffer (40 mM PIPES pH 6.0, 1 mM DTT,20 mg/ml aprotinin, 20 mg/ml leupeptin), and incubated with either PAPbuffer alone or with 1.2 U of PAP (Boehringer-Mannheim, Indianapolis,Ind.), in the presence or absence of phosphatase inhibitors (1 mM sodiumorthovanadate, 100 mM sodium pyrophosphate, 20 mM sodium fluoride) at30° C. for 1 hour, followed by three washes with NP40 lysis buffer.Samples were resolved by SDS-PAGE and immunoblotted with anti-mPALantibody prepared as described above. As a control, PALimmunoprecipitates were treated with either PAP buffer or PAP (1.2 U),as described above.

Example IX Northern Blot Analysis

Northern blots containing 2 μg of poly (A)⁺mRNA isolated from a varietyof murine tissues and embryos were purchased from Clontech.

Where specified, total RNA was prepared from cultured cells using theTRIzol Reagent (Gibco/BRL), as per manufacturers instructions. RNAsamples (10 μg/lane), were separated on formaldehyde-agarose gels andtransferred onto Genescreen nylon membrane (NEN-Dupont, Boston, Mass.),as described by Sambrook et al., supra.

Blots were probed with a 1522 bp AflIII/PstI fragment of the mPAL cDNAwhich was radiolabeled by random hexamer priming (Pharmacia). Blots wereinitially prehybridized for 4 hours at 42° C. in a solution of 50%formamide, 4×SSPE, 1% SDS, 0.5% skim milk powder, 10% dextran sulphateand 10 mg/ml sheared salmon sperm DNA. [³²P]-Radiolabeled probe was thenadded at 10⁶ cpm/ml and the blot further incubated for 16 hours at 42°C. Blots were washed twice for 10 minutes at room temperature in 2×SSC,0.1% SDS, then twice at 65° C. in 0. 1×SSC, 0.1% SDS, and then exposedto film. The blots were also probed with radiolabeled β-actin cDNA(Clontech) or radiolabeled Shc cDNA as an indicator of RNA loading.

Example X In vivo Association Between mPAL and Shc

To determine if mPAL and Shc are associated in vivo, aco-immunoprecipitation assay was performed using the human embryonickidney cell line (293T) (ATCC CRL-1573), co-transfected with pECE-Shcand pcDNA3.1 -mPAL expression vectors described in Example IV above. Thetransfected cells were lysed, immunoprecipitated with a control,non-specific antibody (rabbit anti-mouse IgG), anti-Shc or anti-mPALantibodies, followed by immunoblotting with anti-Shc and anti-mPALantibodies. For this work, the immunoprecipitates were washed threetimes, and the bound proteins resolved via SDS-PAGE. Followingelectrophoretic transfer to membrane, immunoblotting was performed withanti-Shc and anti-mPAL antibodies. This interaction can be disruptedthrough addition of 50 μg of immunizing peptide, which demonstrates thespecificity of this antibody. Typically 2 μg of affinity purifiedanti-mPAL antibody was used for immunoprecipitation.

Neither Shc nor mPAL were nonspecifically immunoprecipitated by thecontrol antibody. The characteristic doublet indicative of p52 and p46Shc was present in the mPAL immunoprecipitate, and likewise, a 75 kDaprotein corresponding to mPAL was detected in the Shc immunoprecipitate,indicating that Shc and mPAL associate in vivo. The presence of mPALimmunizing peptide prevented the immunoprecipitation of mPAL, andconsequently, Shc was no longer observed to co-immunoprecipitate. Thep66 isoform of Shc was also observed to co-immunoprecipitate with mPAL,indicating that the Shc-mPAL interaction is not limited to the p46 andp52 Shc isoforms.

Example XI The SH2 Domain of Shc Specifically Associates with mPAL

To determine which domain of Shc associated with mPAL in vitro,precipitation experiments were performed in which immobilized GST andGST-fusion proteins corresponding to the PTB, CH1 or SH2 domain of Shcwere incubated with lysate from pcDNA3.1- mPAL -transfected 293T cells,and then subjected to immunoblotting with anti-PAL antibody. Thesefusion proteins are discussed in Example m, and these antibodies arediscussed above in Example V. The conditions used are discussed inExample VII. Only the GST-SH2 domain of Shc (GST-Shc SH2), precipitatedmPAL, indicating that this domain was involved in mediating theassociation of Shc with mPAL, in agreement with the data obtained in theyeast-two hybrid system.

To examine the specificity of the mPAL-Shc SH2 interaction, a series ofGST-SH2 fusion proteins generated from several signal transductionmolecules were tested for their ability to precipitate mPAL. A panel ofGST-SH2 fusion proteins generated from several signal transductionmolecules (Shc PTB domain, Blaikie et al., J. Biol. Chem.269:23031-32034(1994), Shc SH2 domain, Pellici et al., Cell 70:93-104(1992), Grb2 SH2 domain, Rozakis-Adcock et al., Nature 360:689-692(1992), Vav SH2 domain Margolis et al., Nature 356: 71-74 (1992),N-terminal SH2 domain of ras-GAP, N-terminal SH2 domain (p85-N) andC-terminal SH2 domain (p85-C) of p85 subunit of PI-3 kinase, N-terminalSH2 domain (PLCγ-N), and C-terminal SH2 domain (PLCγ-C) of phospholipaseC-γ1 Anderson et al., Science 250:979-982 (1992), Ellis et al., Mol.Cell Biol. 12:991-997(1992), and Nck SH2 domain, Lehman et al., Nucl.Acids Res. 18:1048 (1990), were immobilized on glutathione-Sepharosebeads. 5 μg of immobilized GST or GST-fusion protein were individuallyincubated with 293T- mPAL lysate, the beads washed several times, andthe remaining bound proteins resolved via SDS-PAGE, transferred to PVDFmembrane, and immunoblotted with anti-mPAL antibody. None of the othertested GST-SH2 domains were found to bind to mPAL, indicating thatassociation of the Shc SH2 domain with mPAL was specific.

Example XII Interaction of mPAL with the Shc SH2 Domain is Not Dependenton Phosphorylation of mPAL

In the previous experiments, mPAL precipitated by the Shc SH2 domain wasnot detected by immunoblotting with anti-phosphotyrosine (anti-pY),antibodies, suggesting that tyrosine phosphorylation of mPAL is notcritical for Shc SH2 binding.(results not shown) Additionally, theinteraction observed between mPAL and Shc in the yeast-two hybrid systemstrongly suggests that this interaction is independent oftyrosine-phosphorylation (see Example 1). Furthermore, by virtue of itsproduction in E. coli., recombinant GST-mPAL is nottyrosine-phosphorylated and is yet able to precipitate Shc from NIH 3T3lysate, supporting the view that tyrosine-phosphorylation of mPAL is notimportant in the mPAL-Shc SH2 interaction. For this work, 10 μg ofimmobilized recombinant GST, GST-Shc SH2 domain and GST-mPAL fusionprotein were individually incubated with approximately 1 mg of NIH-3T3cell lysate. The beads were washed several times, and the remainingbound proteins resolved via SDS-PAGE, transferred to PVDF membrane, andimmunoblotted with either anti-Shc or anti-mPAL antibodies.

Serine/threonine-phosphorylation-dependent SH2 binding has been reported(Migliaccio et al., EMBO J. 16:706-716 (1997); Malek et al., J. Biol.Chem. 269:33009-33020 (1994); Pendergast et al., Cell 66:161-171(1991)), and thus it was of interest to determine if the mPAL -Shc SH2interaction was phosphorylation-dependent. To investigate this, GST-ShcSH2 was assayed for its ability to associate with dephosphorylated mPAL.Details for this procedure are provided in Example VIII. mPAL wasprecipitated from transfected 293T cells with immobilized GST-Shc SH2,treated with potato-acid phosphatase buffer or potato-acid phosphatase(PAP) to dephosphorylate serine, threonine and tyrosine-phosphorylatedresidues. PAP-treatment of the mPAL immunoprecipitate resulted in adownwards mobility-shift, likely representative of dephosphorylatedmPAL. GST-Shc SH2 was able to precipitate dephosphorylated mPAL,supporting the conclusion that the phosphorylation of mPAL is notcritical for the mPAL-Shc SH2 interaction.

Free phosphotyrosine competed for binding of mPAL to the Shc SH2 domain,whereas free phosphoserine or phosphothreonine did not, implying thatexcess free phosphotyrosine, which is able to occupy thephosphotyrosine-binding pocket of the Shc SH2 domain, preventedinteraction with mPAL. For this work, 2.5 μg of immobilized GST-Shc SH2domain fusion protein was incubated with 293T-mPAL lysate in thepresence of increasing concentrations of free phosphotyrosine,phosphoserine or phosphothreonine (10, 50, and 100 mM). The beads werewashed several times, and the remaining bound proteins resolved viaSDS-PAGE, transferred to PVDF membrane, and immunoblotted with anti-mPALantibody.

To demonstrate that an arginine-to-alanine mutation in the conservedFLVRES, (SEQ ID NO.:6), motif (R397A) in the βB region of the Shc SH2domain, which disrupts its interaction with phosphorylated EGFreceptors, also abrogated binding to mPAL, a Shc SH2 domain mutant (SH2R 397A)), which is unable to bind to phosphotyrosine-containingsubstrates (Arg397 mutated to Ala), was generated. The Shc2 (R297A)mutein protein and the wildtype Shc SH2 domain (SH2), were expressed asGST-fusion proteins, and immobilized onto glutathione-Sepharose beads(see Example III above). 5 μg of GST or GST-fusion protein wereindividually incubated with 293T-mPAL lysate, the beads washed severaltimes, and the remaining bound proteins resolved via SDS-PAGE,transferred to PVDF membrane, and immunoblotted with anti-mPAL antibody.These studies showed that the Shc SH2 mutant protein R397A not onlyprevented the binding of the mutant protein to phosphorylated EGFreceptors, but also abrogated the binding of the Shc SH2 mutant proteinto mPAL. This suggests that the mPAL-Shc SH2 interaction may involvestructural elements similar to those involved in SH2phosphotyrosine-peptide interactions.

The mechanism of SH2 binding is novel. Unlike other SH2domain-phosphopeptide interactions, the mPAL-Shc SH2 interaction appearsto be independent of phosphorylation. However, certain structuralelements involved in Shc SH2-phosphotyrosine peptide interactions arelikely involved in the mPAL-Shc SH2 domain interaction, since occupancyof the phosphotyrosine-binding pocket with free phosphotyrosine, ormutation of the conserved R397 residue involved in phosphotyrosinebinding disrupts the mPAL-Shc SH2 domain interaction. It is possiblethat binding of mPAL to the Shc SH2 domain is mediated by the acidicregions in mPAL because phosphotyrosine independent interactions betweenSH2 domains of other proteins such as BCR-ABL, Lck and Blk and sequencesrich in serine and glutamic acid rich have been previously reported(Joung et al., Proc. Natl. Acad. Sci. USA 93:5991-5995 (1996); Malek etal., J. Biol. Chem. 269:33009-33020 (1994); Pendergast et al., Cell66:161-171 (1991)). In some cases, serine phosphorylation has been shownto be important for binding (Malek et al. J. Biol. Chem. 269:33009-33020(1994); Pendergast et al., Cell 66:161-171 (1991)). This does not appearto be the case for the mPAL-Shc protein interaction since phosphatasetreatment of mPAL does not abrogate binding, nor is the interactioncompeted by free phosphoserine or phosphothreonine. The exact nature ofthe Shc SH2-binding site of mPAL is yet to be elucidated.

Example XIII Expression of mPAL Correlates with Cellular Proliferation

In order to assess whether expression of mPAL correlates with cellularproliferation and/or development multiple tissue Northern blots of polyA⁺RNA extracted from adult murine tissues were purchased from Clontech.A [³²P]-labeled 1.5 kb AflIII/PstI fragment of mPAL cDNA was utilized asprobe. Molecular weight standards were indicated on the membrane. Themembrane was exposed to X-ray film overnight at −80° C. Equal loading ofmRNA was confirmed by reprobing the membrane with [³²P]-labeled β-actincDNA probe.

Northern blot analysis of the multiple tissue RNA blot revealed a singlemPAL RNA transcript of less than 2.4 kb in size, in close agreement withthe cDNA size of 2.2 kb. mPAL RNA was strongly expressed in the testis,but was present at a much lower level in spleen, lung and heart. mPALRNA was, however, absent from brain, liver, and skeletal muscle. Indeveloping embryos mPAL RNA expression was detected at all stages ofembryonic development. This pattern of expression suggested that mPALexpression is restricted to tissues containing a proliferating cellpopulation, but is absent from quiescent tissues. The low levels of mPALRNA detected in murine lung and heart may result from tissuecontamination with activated lymphocytes, which we have demonstrated,express high levels of mPAL.

In agreement with data obtained by Northern blot analysis, mPAL proteinwas expressed in mouse spleen, testis and thymus, all of which containproliferating cells, but was absent from normal quiescent tissues. Awide variety of murine tissues were homogenized in NP40 lysis bufferincluding brain, heart, kidney, liver, lung, skeletal muscle, pancreas,spleen, testis, thymus, and lymphocytes. Immunoprecipitations of mPALwas performed on 1 mg of total protein from each of these tissues usingthe same techniques as described for cell lines (Example VI). mPALprotein was expressed in mouse spleen, testis, and thymus, each of whichrepresents a tissue containing actively dividing cells. mPAL was notdetectable in other adult tissues which do not normally proliferate invivo. In addition, it was determined that mPAL protein levels areelevated in all murine cell lines tested to date, provided that thecells are actively proliferating.

A human tumor cell line Northern blot (purchased from Clonetech), wasprobed with murine PAL cDNA as previously described Example IX). TheNorthern blot contained poly (A)⁺RNA from the following cell lines:HL-60 promyelocytic leukemia) (ATCC CCL-240), HeLa Cell S3 (cervicalcarcinoma) (ATCC CCL-2.2), K562 (chronic myelogenous leukemia) (ATCCCCL-243), MOLT-4 (acute lymphoblastic leukemia) (ATCC CRL-1552), RajiBurkitt's lymsphoma) (ATCC CCL-86), SW480 (colorectal carcinoma) (ATCCCCL-228), A549 (lung carcinoma) (ATCC CCL-185) and G361 (malignantmelanoma) (ATCC CRL-1424). Elevated levels of human PAL RNA weredetected in all the tumor cell lines.

mPAL protein and mRNA were also elevated in tissues containingproliferating cells and in proliferating cell lines, but were absent innormal, quiescent tissues and growth-arrested cells. Factors whichstimulate cell cycle progression and proliferation also stimulate mPALexpression, whereas factors which inhibit cellular proliferation,including serum withdrawal, contact inhibition, and terminaldifferentiation, inhibit the expression of mPAL. Within 24 hoursfollowing transfer to a culture medium containing low concentrations ofserum (DMEM containing 0.5% fetal bovine serum), NIH/3T3 cells aredepleted of both mPAL mRNA (as detected by northern blot analysis), andprotein (as detected by immunoprecipitation and Western blot analysis).Cells plated at subconfluent levels express both mPAL protein and RNA.However, within 24 hours of reaching confluence, cells are depleted ofmPAL protein and RNA (see Example XV below). At this stage the cellshave become post-mitotic.

These data support a role for mPAL in cell proliferation. mPAL may beinvolved in progression through the cell cycle rather than the immediateearly response because mPAL mRNA is expressed during entry into S phaseand passage through G2/M. Preliminary data from cyclohexamide treatedcells also suggests that mPAL is not an immediate early response gene.Proteins whose expression can be induced by growth factors in theabsence of any de novo protein synthesis are considered to be encoded byearly response genes. Resting cells (cells in G0), will express earlyresponse genes typically within one hour (often within minutes),following stimulation by growth factors. Many early response genesencode transcription factors required for induction of delayed responsegenes.

To test if a gene is an immediate early response gene, cells arestimulated in the presence of an inhibitor of protein synthesis, such ascyclohexamide. If the mRNA for the protein is detectable, even in theabsence of protein synthesis, then this protein is encoded by an earlyresponse gene.

Because mPAL RNA and protein are not detected until cells entering Sphase (12-16 hours following stimulation by growth factor), it is alsounlikely that mPAL is not an early response gene.

Overall, the pattern of expression of mPAL protein and RNA support arole for mPAL in signaling pathways governing cellular proliferation.

Example XIV Induction of mPAL by Addition of Exogenous Growth Factors

For the examples discussed below, unless otherwise indicted, NIH 3T3,P19, and 293T cells were cultured in Dulbecco's Modified Eagle medium(DMEM) supplemented with 10% fetal bovine serum (Sigma), 200 mML-glutamine, 5×10⁻⁵ M β-mercaptoethanol, 5 U/ml penicillin C, and 5μg/ml streptomycin sulfate.

In order to characterize the serum mediated induction of mPAL, NIH 3T3cells were grown to 70-80% confluence, washed twice in phosphatebuffered saline (PBS) and then transferred to media containing 0.5%fetal bovine serum for 48 hours to achieve quiescence. Serum starvedcells were then transferred to media containing 20% fetal bovine serum.Samples were harvested for mPAL protein or mPAL RNA isolation at timeperiods of 0, 1, 4, 8, 12, and 24 hours. Cells isolated at each timepoint were stained with propidium iodide and subjected to cell cycleanalysis by flow cytometry. (Jacoby et al., eds., supra).

In total RNA isolated from serum starved NIH/3T3, mPAL RNA wasdetectable at low levels 1 hour following stimulation. Levels remainedlow for 8 hours following addition of serum. Expression, however,increased detectably by 12 hours through 24 hours where RNA levels werehigh. Detectable protein expression appeared to lag behind RNA levels,as mPAL protein is only detectable at the 24 hour time point. For thiswork, at each time point, one plate of NIH/3T3 was NP40 lysed. The mPALprotein was immunoprecipitated from 1 mg of cell lysate as describedabove. Immunoprecipitated proteins were separated by SDS-PAGE,transferred to Immobilon membrane and immunoblotted with anti-mPALantibodies. Control immunoprecipitates were prepared from equal amountsof protein. In addition, 10 μg of whole cell lysate was alsoimmunoblotted for Shc.

Cell cycle analysis demonstrated a well synchronized passage of thesecells through the cell cycle following addition of fetal calf serum.Cells remained primarily in G0/G1 through 8 hours of stimulation,entered S-phase by 12 hours, and 20% of the cells were in G2M at 24hours following addition of fetal calf serum. Taken together, these dataindicate that mPAL is low or absent in cells in G0/G1, but is elevatedin cells committed to cell cycle progression, or actively cycling cells.

Example XV Expression of mPAL is Down-regulated in Cell Lines by ContactInhibition and Terminal Differentiation

Since mPAL mRNA and protein levels appeared to correlate with cellproliferation in tissues, levels of mRNA were analyzed in proliferatingversus contact inhibited NIH/3T3 cells in culture. NIH/3T3 cells wereplated at approximately 50% confluence in DMEM supplemented with 10%fetal bovine serum, and allowed to proliferate over several days. Levelsof mPAL mRNA were measured by Northern blot analysis and evaluatedrelative to cell confluence. For this work, NIH/3T3 cells were plated ata density of approximately 50% confluence. The mRNA was harvested fromthese cells over five consecutive days, and the percentage confluence ofeach plate was noted at the time of harvest. The mRNA samples (10μg/lane), were separated on formaldehyde-agarose gels and transferredonto Genescreen nylon membrane. Blots were probed for mPAL mRNA asdescribed above. This blot was exposed to X ray film at −80° C. for 2days. Equal loading of mRNA was confirmed by reprobing the membrane forShc which remains constant upon contact inhibition. Actively growing,subconfluent NIH/3T3 cells expressed relatively high levels of mPALmRNA. However, within 24 hours following cultures reaching confluence,levels of mPAL mRNA decreased to undetectable levels. In contrast, ShcmRNA levels remained constant throughout the time course, irrespectiveof cell density.

Levels of mPAL were also measured in the embryonic carcinoma cell lineP19 in response to differentiation inducing agents. The embryoniccarcinoma cell line, P19, can be induced to differentiate to a neuralphenotype by incubation in the presence of retinoic acid, and to amuscle phenotype in the presence of DMSO (McBurney, Int. J. Dev. Biol.37:35-140 (1993)). Following 6 to 7 days of treatment with retinoicacid, as many as 85% of P19 cells expressed neuronal markers and becomepost-mitotic (McBurney et al., J. Neurosci. 8:1063-1073 (1988);McBurney, Int. J. Dev. Biol. 37:35-140 (1993)). Additionally, DMSOtreated P19 cells differentiated towards mesodermal and endodermallineages. Approximately 25% cardiac muscle cells are observed following6-7 days of DMSO treatment (McBurney, Int. J. Dev. Biol. 37:35-140(1993); Rudnicki et al. J. Cell Physio. 142:89-98 (1990)), while theremainder of cells in culture continue to grow and differentiate intoskeletal muscle as well as in to other less well defined cell types(McBurney et al., Int. J. Dev. Biol. 37:35-140 (1993)).

Levels of mPAL protein were measured by immunoprecipitation and Westernblot analysis in P19 cells induced to differentiate in the presence ofretinoic acid or DMSO. For this work, the pluripotent murine embryoniccarcinoma cell line, P19, was induced to differentiate into aneuronal/glial phenotype and into muscle-like phenotype in the presenceof retinoic acid and DMSO, as discussed above. The mPAL protein wasimmunoprecipitated from 1 mg of NP-40 cell lysate from each sample.Immunoprecipitates were separated by SDS-PAGE, transferred to Immobilonand immunoblotted with anti-mPAL. In control experiments, 25 μg of wholecell lysate was subjected to SDS-PAGE and Western blot analysis with 0.5μg anti-β tubulin antibody/mL of blotting solution as probe to controlfor equal loading. While the parental, rapidly proliferating P19 cellsexpressed mPAL protein, DMSO differentiated P19 cells expressedsignificantly lower levels of mPAL. Furthermore, in P19 cells treatedfor 7 days in the presence of retinoic acid, mPAL is virtuallyundetectable. Levels of β-tubulin remain constant throughoutdifferentiation. These data further support the hypothesis that mPALexpression is restricted to proliferating cells and is down regulatedupon growth inhibition.

Example XVI T-cell Activation Results in mPAL Expression and Formationof a mPAL-Shc Complex

The restricted expression pattern of mPAL suggested that the associationof She and mPAL in vivo might be limited to specific cell types or tocells which have been induced to proliferate and therefore express highlevels of mPAL protein. Since the proliferation rate of primary T cellsis readily regulated in vitro, the levels of mPAL expression and itsassociation with She were investigated in resting versus activated Tcells. Primary murine T cells were isolated from wild type and CTLA4deficient mice (Walterhouse et al., Science 270:985-988 (1995);Marengere et al., Science 272:1170-1173 (1996)). Wild type murine Tcells were activated by cross-linking the T cell receptor on anti-CD3coated tissue culture plates essentially as described in Marengere etal., J. Immunol. 159:70-76 (1997).

Primary mouse lymphocytes, activated by cross linking CD3, expressedhigh levels of mPAL, while unstimulated cells did not. For this work,T-cells isolated from wild type mice were stimulated by cross-linkingthe T-cell receptor with anti-CD3 antibodies. Samples were collectedover 4 days and lysed in NP-40 lysis buffer. The mPAL protein wasimmunoprecipitated from 1 mg of lysate from each sample, and theimmunoprecipitates were resolved via SDS-PAGE. Following electrophoretictransfer to PVDF membrane, immunoblotting was performed with anti-mPALantibodies. The blot was then stripped and immunoblotted with anti-Shcantibodies. The timing of the expression of mPAL was coincident withT-cell activation and proliferation. Shc immunoblots of anti-mPALimmunoprecipitates revealed the coprecipitation of Shc with mPALfollowing T cell activation and concurrent with mPAL expression.Furthermore, constitutively activated primary T-cells isolated from aCTLA4 deficient mouse (Marengere et al., Science 272: 1170-1173 (1996);Waterhouse et al., Science 270:985-988 (1995)), expressed mPAL proteinwhich coprecipitated with Shc, while mPAL was undetectable in T cellsisolated from a wild type mouse.

Example XVII Cloning of Human PAL (hPAL)

A. Library Screen

A [³²P]-labeled 1.5 kb AflIII/PstI fragment of mouse PAL (mPAL) cDNA wasused to probe a human HeLa 5′-STRETCH cDNA Library which was purchasedfrom Clontech. The cDNA fragment was separated by 1% agarose gelelectrophoresis (as described in Sambrook et al., Supra). TheAflIII/PstI fragment was excised from the gel and the cDNA isolatedusing Qiaex II gel Extraction Kit (Qiagen). 50 ng of the cDNA fragmentwas labeled with ³²P by random hexamer priming using the Pharmacia T7QuickPrime Kit (Pharmacia). Filters were initially prehybridized for 4hours at 42° C. in a solution of 50% formamide, 4×SSPE, 1% SDS, 0.5%skim milk powder, 10% dextran sulphate and 1 mg/mL sheared salmon spermDNA. [³²P]-Radiolabeled probed was then added at 1×10⁶ CPM/ml and theblot further incubated for 16 hours at 42° C. Filters were washed twicefor 10 minutes at room temperature in 2×SSC, 0.1% SDS, then twice for 20minutes at 56° C. in 0.1×SSC, 0.1% SDS, then exposed to film.

The cDNA sequence of largest of the clones (clone 7.1.1), isolated fromthe HeLa library screen was utilized to design primers for 5′ and 3′RACE in order to obtain the full length cDNA sequence for hPAL fromMarathon Ready HeLa cDNA (Clontech).

B. 5′ and 3′ RACE Conditions

5′ and 3′ RACE was carried out as a two reaction process using two setsof primers. (Frohman et al., supra). 1. 5′ RACE Primers:

Outer primer: 5′-CCTCAGGGACCTTGCAGTCAGCTA-3′ (SEQ ID NO.:7)

Nested primer: 5′-CTTTCTCCAGAGACGCCAGCTCCT-3′ (SEQ ID No.:8)

2. 3′ RACE Primers

Outer primer: 5′-GACTACGCTGGAAAACTGTGTGCT-3′ (SEQ ID NO.:9)

Nested primer: 5′-ATGGGATCCATCACTGCAAGGAAG-3′ (SEQ ID NO.:10)

3. RACE Reaction Mixture:

“Expand High Fidelity PCR System” (Boehringer Mannheim, Indianapolis,Ind.) polymerase and 10× reaction buffer was used for PCR reactions.

Primary PCR reaction (50 ul final volume) Marathon ready cDNA 5 ul AP1primer (10 uM) 1 ul Outer primer (10 uM) 1 ul dNTP (2.5 mM) 4 ul 10 XPCR buffer 5 ul Taq polymerase 0.75 ul dH2O 33 ul

Secondary PCR reaction (50 μl final volume) 1/50 dilution of Primaryreaction 5 μl AP2 primer (10 uM) 1 μl nested primer (10 uM) 1 μl dNTP(2.5 mM) 4 μl 10 X PCR buffer 5 μl Taq polymerase 0.75 μl dH2O 33 μl

PCR Program:

1. 3 minutes at 94° C.

2. 1 minute at 94° C.

3. 1.5 minutes at 56° C.

4. 3 minutes at 72° C.

5. Return to step 2 for 29 cycles

6. 10 minutes at 72° C.

7. Indefinitely at 4° C. (end of program)

PCR products were gel purified and cloned into pCR2. 1 using the TAcloning system purchased from Invitrogen. The full length human cDNA wasconstructed, using conventional cloning techniques, as a composite fromsequences obtained from 3′ and 5′ RACE and the cDNA clone 7.1.1originally isolated from the HeLa cDNA library, The full length clonewas subsequently subcloned into pBluescript cloning vector (Stratagene)

The foregoing examples are presented by way of illustration and are notintended to limit the invention as set out in the appended claims.

12 2581 base pairs nucleic acid single linear cDNA /desc = “hPAL cDNA” 1GGATCCGCGG GAAATTTGAA ATGGCTGACG GGTCGCTGAC GGGCGGCGGT CTGGAGGCAG 60CGGCCATGGC GCCGGAGCGC ACGGGCTGGG CGGTGGAGCA GGAGCTGGCG TCTCTGGAGA 120AAGGTTTGTT CCAAGATGAA GATTCATGCA GTGATTGTAG CTACCGTGAT AAACCAGGTT 180CTAGTTTACA AAGTTTTATG CCAGAAGGAA AAACCTTTTT CCCAGAAATT TTCCAAACAA 240ATCAACTTTT GTTCTATGAG CGATTCAGAG CCTATCAAGA TTACATTTTA GCTGACTGCA 300AGGCCTCTGA GGTACAGGAA TTCACAGCTG AGTTCTTGGA GAAGGTCCTT GAGCCATCTG 360GATGGCGGGC AGTCTGGCAC ACTAATGTGT TCAAGGTGCT GGTTGAGATC ACAGATGTGG 420ACTTTGCAGC CTTGAAGGCA GTGGTGAGGC TTGCTGAACC ATACCTCTGT GACTCTCAAG 480TGAGCACTTT TACCATGGAG TGCATGAAGG AGCTCCTTGA TCTGAAGGAG CATCGGTTGC 540CCCTGCAGGA GCTGTGGGTG GTGTTTGATG ATTCAGGAGT GTTTGACCAG ACAGCCCTTG 600CAATTGAGCA TGTCAGATTT TTCTACCAAA ACATTTGGAG GAGTTGGGAT GAAGAAGAGG 660AGGATGAATA CGATTATTTT GTCAGATGTG TTGAACCTCG ATTAAGATTG CATTATGACA 720TTCTTGAAGA CCGAGTTCCA TCAGGACTTA TTGTTGACTA CCACAATCTG TTGTCTCAAT 780GTGAGGAGAG TTACAGGAAA TTTTTAAATC TGAGAAGCAG TTTGTCAAAT TGTAACTCTG 840ATTCCGAGCA GGAAAATATC TCCATGGTGG AAGGGTTAAA ATTGTATTCG GAGATGGAAC 900AGTTGAAACA AAAGCTGAAA CTCATTGAGA ATCCTTTGTT GAGGTATGTG TTTGGTTATC 960AGAAGAATTC TAACATCCAA GCAAAGGGTG TCCGTTCCAG CGGTCAGAAG ATCACTCATG 1020TGGTCTCCTC CACCATGATG GCTGGTCTCC TGCGGTCCCT GCTTACGGAC AGGCTTTGCC 1080AGGAGCCTGG TGAGGAAGAA AGAGAAATTC AGTTCCATAG TGATCCATTG TCTGCTATAA 1140ATGCCTGCTT CGAAGGTGAC ACTGTTATTG TTTGTCCTGG CCATTATGTG GTACATGGCA 1200CTTTCTCCAT TGCTGACTCC ATTGAGTTGG AAGGATATGG CCTACCAGAT GACATTGTGA 1260TAGAAAAGAG GGGCAAAGGC GACACTTTTG TGGACTGCAC TGGTGCTGAT ATTAAAATCT 1320CAGGCATAAA ATTTGTTCAG CATGATGCTG TAGAGGGAAT CTTAATTGTT CACCGTGGTA 1380AGACTACGCT GGAAAACTGT GTGCTGCAGT GTGAGACGAC CGGAGTCACA GTGCGGACAT 1440CAGCAGAGTT TCTAATGAAG AACTCGGATT TATATGGCGC CAAGGGTGCT GGTATAGAAA 1500TCTACCCTGG GAGTCAGTGC ACCCTGAGTG ACAATGGGAT CCATCACTGC AAGGAAGGGA 1560TCCTCATTAA GGACTTCTTA GATGAACATT ATGACATTCC CAAGATATCC ATGGTGAATA 1620ATATAATACA TAATAATGAA GGTTATGGTG TTGTCTTGGT GAAACCTACA ATCTTCTCTG 1680ACCTGCAAGA AAATGCTGAA GATGGAACTG AAGAAAATAA AGCGCTTAAA ATTCAGACAA 1740GTGGAGAGCC AGATGTGGCT GAAAGAGTGG ATCTAGAGGA GCTGATTAAG TGTGCAACTG 1800GTAAAATGGA GCTTTGTGCA AGAACTGACC CTTCTGAGCA AGTCGAGGGA AATTGTGGAA 1860TTGTAAATGA ACTAATTGCT GCCTCCACAC AGAAAGGCCA GATAAAGAAG AAAAGGTTGA 1920GTGAACTGGG GATCACGCAA GCTGATGACA ACTTAATGTC ACAGGAGATG TTTGTTGGGA 1980TTGTGGGGAA CCAGTTCAAG TGGAATGGGA AAGGTAGTTT TGGCACATTT CTTTTCTGAC 2040TACAGTGATG CAAGTAGATA GCAAAATACT GGATTTTGCA CATGCTGCCC TAAGAATCAC 2100TGCTGCCATT GTAGTTTGCT GTATTGTCTG TATTTTATAT TTGATTATTT GGGCTTGAGT 2160GAAAGGTAGA TTTATTTCCA TTTGCAGGTG TTGCACATAA AACACTCCCT CTTTATAAGA 2220AAAATCATAA ATGCATATAA AATAGAAAAT ATTTGGAGAT TGCTTATCTG AAAGTCTTGC 2280TTTCTTATAC ACATGGTTCT CTCATATTAA GCCTGGTGGT AACTTTTTAG TGTAATTACC 2340TTTAGCACTT CAAAGACGAG GAAGTAAGGA AGGGAATGCA AGACTAGTGC ATAAAAATGC 2400AATAGGTGTC ATATGTACAG CATTCTTCTT AGAGTTGCCT TTTCATCCCA ATTACAGTGA 2460GTCTGATTTC CATCCTGTAT TTGCATAATA CTTGTCTTAA AATAAAAGCT TTTATGATTG 2520GGGAAAAAAA AAAAAAAAAA GGAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2580 A2581 672 amino acids amino acid Not Relevant linear peptide /desc =“hPAL peptide” 2 Met Ala Asp Gly Ser Leu Thr Gly Gly Gly Leu Glu Ala AlaAla Met 1 5 10 15 Ala Pro Glu Arg Thr Gly Trp Ala Val Glu Gln Glu LeuAla Ser Leu 20 25 30 Glu Lys Gly Leu Phe Gln Asp Glu Asp Ser Cys Ser AspCys Ser Tyr 35 40 45 Arg Asp Lys Pro Gly Ser Ser Leu Gln Ser Phe Met ProGlu Gly Lys 50 55 60 Thr Phe Phe Pro Glu Ile Phe Gln Thr Asn Gln Leu LeuPhe Tyr Glu 65 70 75 80 Arg Phe Arg Ala Tyr Gln Asp Tyr Ile Leu Ala AspCys Lys Ala Ser 85 90 95 Glu Val Gln Glu Phe Thr Ala Glu Phe Leu Glu LysVal Leu Glu Pro 100 105 110 Ser Gly Trp Arg Ala Val Trp His Thr Asn ValPhe Lys Val Leu Val 115 120 125 Glu Ile Thr Asp Val Asp Phe Ala Ala LeuLys Ala Val Val Arg Leu 130 135 140 Ala Glu Pro Tyr Leu Cys Asp Ser GlnVal Ser Thr Phe Thr Met Glu 145 150 155 160 Cys Met Lys Glu Leu Leu AspLeu Lys Glu His Arg Leu Pro Leu Gln 165 170 175 Glu Leu Trp Val Val PheAsp Asp Ser Gly Val Phe Asp Gln Thr Ala 180 185 190 Leu Ala Ile Glu HisVal Arg Phe Phe Tyr Gln Asn Ile Trp Arg Ser 195 200 205 Trp Asp Glu GluGlu Glu Asp Glu Tyr Asp Tyr Phe Val Arg Cys Val 210 215 220 Glu Pro ArgLeu Arg Leu His Tyr Asp Ile Leu Glu Asp Arg Val Pro 225 230 235 240 SerGly Leu Ile Val Asp Tyr His Asn Leu Leu Ser Gln Cys Glu Glu 245 250 255Ser Tyr Arg Lys Phe Leu Asn Leu Arg Ser Ser Leu Ser Asn Cys Asn 260 265270 Ser Asp Ser Glu Gln Glu Asn Ile Ser Met Val Glu Gly Leu Lys Leu 275280 285 Tyr Ser Glu Met Glu Gln Leu Lys Gln Lys Leu Lys Leu Ile Glu Asn290 295 300 Pro Leu Leu Arg Tyr Val Phe Gly Tyr Gln Lys Asn Ser Asn IleGln 305 310 315 320 Ala Lys Gly Val Arg Ser Ser Gly Gln Lys Ile Thr HisVal Val Ser 325 330 335 Ser Thr Met Met Ala Gly Leu Leu Arg Ser Leu LeuThr Asp Arg Leu 340 345 350 Cys Gln Glu Pro Gly Glu Glu Glu Arg Glu IleGln Phe His Ser Asp 355 360 365 Pro Leu Ser Ala Ile Asn Ala Cys Phe GluGly Asp Thr Val Ile Val 370 375 380 Cys Pro Gly His Tyr Val Val His GlyThr Phe Ser Ile Ala Asp Ser 385 390 395 400 Ile Glu Leu Glu Gly Tyr GlyLeu Pro Asp Asp Ile Val Ile Glu Lys 405 410 415 Arg Gly Lys Gly Asp ThrPhe Val Asp Cys Thr Gly Ala Asp Ile Lys 420 425 430 Ile Ser Gly Ile LysPhe Val Gln His Asp Ala Val Glu Gly Ile Leu 435 440 445 Ile Val His ArgGly Lys Thr Thr Leu Glu Asn Cys Val Leu Gln Cys 450 455 460 Glu Thr ThrGly Val Thr Val Arg Thr Ser Ala Glu Phe Leu Met Lys 465 470 475 480 AsnSer Asp Leu Tyr Gly Ala Lys Gly Ala Gly Ile Glu Ile Tyr Pro 485 490 495Gly Ser Gln Cys Thr Leu Ser Asp Asn Gly Ile His His Cys Lys Glu 500 505510 Gly Ile Leu Ile Lys Asp Phe Leu Asp Glu His Tyr Asp Ile Pro Lys 515520 525 Ile Ser Met Val Asn Asn Ile Ile His Asn Asn Glu Gly Tyr Gly Val530 535 540 Val Leu Val Lys Pro Thr Ile Phe Ser Asp Leu Gln Glu Asn AlaGlu 545 550 555 560 Asp Gly Thr Glu Glu Asn Lys Ala Leu Lys Ile Gln ThrSer Gly Glu 565 570 575 Pro Asp Val Ala Glu Arg Val Asp Leu Glu Glu LeuIle Glu Cys Ala 580 585 590 Thr Gly Lys Met Glu Leu Cys Ala Arg Thr AspPro Ser Glu Gln Val 595 600 605 Glu Gly Asn Cys Glu Ile Val Asn Glu LeuIle Ala Ala Ser Thr Gln 610 615 620 Lys Gly Gln Ile Lys Lys Lys Arg LeuSer Glu Leu Gly Ile Thr Gln 625 630 635 640 Ala Asp Asp Asn Leu Met SerGln Glu Met Phe Val Gly Ile Val Gly 645 650 655 Asn Gln Phe Lys Trp AsnGly Lys Gly Ser Phe Gly Thr Phe Leu Phe 660 665 670 2246 base pairsnucleic acid single linear cDNA /desc = “mouse PAL cDNA” 3 GTAAATTTGAAATGGCTGAT GATTTGCGGG CTGGTGGAGT TCTGGAACCT ATAGCTATGG 60 TTCCACCGAGACCTGACTTG GCGGCGGAGA AGGAACCGGC GTCCTGGAAG GAAGGTTTAT 120 TCTTGGATGCAGATCCATGC AGTGATCAAG GCTATCATGC TAATCCAGGT GCTACTGTAA 180 AAACTCTCATACCAGAAGGA AAAACTCCTT TTCCACGAAT TATCCAAACA AATGAACTTC 240 TGTTTTATGAACGATTCAGA GCCTATCAAG ATTACATTTT AGCTGACTGT AAGGCCTCTG 300 AGGTAAAGGAATTCACAGTC AGCTTCTTGG AAAAGGTCCT TGAACCATCT GGATGGTGGG 360 CAGTCTGGCACACTAATGTG TTTGAGGTGT TGGTTGAGGT TACAAATGTG GACTTTCCAT 420 CCCTGAAGGCGGTCGTAAGG CTTGCAGAGC CATGCATCTA TGAATCTAAA TTGAGCACGT 480 TTACCTTGGCCAATGTGAAG GAGCTTTTGG ACCTGAAGGA GTTTCATCTG CCTCTGCAGG 540 AGTTGTGGGTGGTATCAGAT GACTCACATG AATTCCACCA GATGGCACTT GCAATTGAGC 600 ACGTCAGATTTTTCTACAAA CACATCTGGA GGAGTTGGGA TGAGGAAGAG GAGGATGAGT 660 ATGACTATTTTGTCAGATGT GTTGAACCTC GACTGAGATT GTATTATGAC ATACTTGAAG 720 ATCGAGTTCCCTCGGGACTT ATTGTTGACT ACCACAATCT GTTGTCTCAA TGTGAAGAGA 780 GTTACAGGAAATTTTTAAAT CTGAGAAGCA GTTTGTCCAA TTGTAATTCT GATTCTGAGC 840 AGGAAAATATCTCCATGGTG GAAGGGTTAA ATTTGTATTC AGAAATTGAA CAGTTGAAAC 900 AAAAGCTAAAGCTCATTGAG AATCCTTTGT TAAGATATGT TTTTGGTTAT CAGAAGAACT 960 CTAATATCCAAGGAAAGGGT ACTCGTCAAA ATGGCCAGAA GGTCATCCAT GTGGTTTCCT 1020 CCACCATGAAGACAGGTCTA CTTCGGTCTC TATTCAAGGA CAGGTTTTGT GAGGAGTCTT 1080 GCAAAGAAGAAACAGAAATT AAGTTCCATA GTGATCTGTT GTCTGGTATA AATGCCTGCT 1140 ATGATGGTGACACTGTCATT ATTTGTCCTG GCCATTATGT AGTTCATGGC ACCTGTTCCA 1200 TAGCTGACTCCATTGAGTTG GAAGGATATG GCCTACCAGA TGACATTGTC ATAGAAAAGA 1260 GGGGCAAAGGAGATACTTTT GTGGATTGCA CGGGTATGGA TGTTAAAATT TCAGGCATAA 1320 AATTTATTCAGCATGATTCT GTGGAAGGAA TCTTAATCAT TCACCATGGC AAGACCACAC 1380 TGGAAAACTGTGTACTACAA TGTGAAACCA CAGGAGTCAC AGTGCGCACA TCAGCAGAAC 1440 TTTTCATGAAAAACTCAGAT GTATATGGTG CCAAGGGTGC TGGTATAGAA ATATATCCTG 1500 GAAGTAAATGTACCCTGACT GACAATGGAA TCCATCACTG CAAGGAAGGA ATTCTCATTA 1560 AGGACTTCCTTGATGAACAT TATGATATTC CCAAAATATC GATGATAAAT AACGTCATAC 1620 ACAATAATGAAGGTTATGGT GTTGTTTTGG TGAAGCCTAC AATTTTCTGT GATCTACAGG 1680 AAAATACACAAGATGAAATT AATGACAATA TGGTTCAGAA AAATAAAGAG GCAGATGTCA 1740 CTGAAGGATTAGATCTGGAA GAAATGCTTC AGTGTGTGGC TAGCAAAATG GAGCCTTATG 1800 CCACAGCTGACTTTAATGAA CAAGCTAAGG GAAACTGTGA AATTATAAAT GAACTACTTG 1860 CTATTTCCATGCAAAAAGGC CGGATGAAGA AAAGACTGAG TGAACTTGGG ATTACACAAG 1920 CTGATGACAACATAATGTCA CAGGAGATGT TTATTGAAAT TATGGGGAAC CAGTTTAAGT 1980 GGAATGGCAAAGGGAGTTTT GGCACATTTC TTTACTAGCT ACAATAATAT CAATACTCAC 2040 AAAATACTGTATTTTGAACA TGTCTTAAGT ATGCTGCTTA TATACTTTGC TTCATTTGCT 2100 TCATGGCTGTGTATTATATA AAGTGTACTT GACCAAAAAA AAAAAAAAAA AAAAAAAAAA 2160 AAAAAAAAAAAAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2220 AAAAAAAAAAAAAAAAAAAA AAAAAA 2246 668 amino acids amino acid Not Relevant linearpeptide /desc = “mouse PAL peptide” 4 Met Ala Asp Asp Leu Arg Ala GlyGly Val Leu Glu Pro Ile Ala Met 1 5 10 15 Val Pro Pro Arg Pro Asp LeuAla Ala Glu Lys Glu Pro Ala Ser Trp 20 25 30 Lys Glu Gly Leu Phe Leu AspAla Asp Pro Cys Ser Asp Gln Gly Tyr 35 40 45 His Ala Asn Pro Gly Ala ThrVal Lys Thr Leu Ile Pro Glu Gly Lys 50 55 60 Thr Pro Phe Pro Arg Ile IleGln Thr Asn Glu Leu Leu Phe Tyr Glu 65 70 75 80 Arg Phe Arg Ala Tyr GlnAsp Tyr Ile Leu Ala Asp Cys Lys Ala Ser 85 90 95 Glu Val Lys Glu Phe ThrVal Ser Phe Leu Glu Lys Val Leu Glu Pro 100 105 110 Ser Gly Trp Trp AlaVal Trp His Thr Asn Val Phe Glu Val Leu Val 115 120 125 Glu Val Thr AsnVal Asp Phe Pro Ser Leu Lys Ala Val Val Arg Leu 130 135 140 Ala Glu ProCys Ile Tyr Glu Ser Lys Leu Ser Thr Phe Thr Leu Ala 145 150 155 160 AsnVal Lys Glu Leu Leu Asp Leu Lys Glu Phe His Leu Pro Leu Gln 165 170 175Glu Leu Trp Val Val Ser Asp Asp Ser His Glu Phe His Gln Met Ala 180 185190 Leu Ala Ile Glu His Val Arg Phe Phe Tyr Lys His Ile Trp Arg Ser 195200 205 Trp Asp Glu Glu Glu Glu Asp Glu Tyr Asp Tyr Phe Val Arg Cys Val210 215 220 Glu Pro Arg Leu Arg Leu Tyr Tyr Asp Ile Leu Glu Asp Arg ValPro 225 230 235 240 Ser Gly Leu Ile Val Asp Tyr His Asn Leu Leu Ser GlnCys Glu Glu 245 250 255 Ser Tyr Arg Lys Phe Leu Asn Leu Arg Ser Ser LeuSer Asn Cys Asn 260 265 270 Ser Asp Ser Glu Gln Glu Asn Ile Ser Met ValGlu Gly Leu Asn Leu 275 280 285 Tyr Ser Glu Ile Glu Gln Leu Lys Gln LysLeu Lys Leu Ile Glu Asn 290 295 300 Pro Leu Leu Arg Tyr Val Phe Gly TyrGln Lys Asn Ser Asn Ile Gln 305 310 315 320 Gly Lys Gly Thr Arg Gln AsnGly Gln Lys Val Ile His Val Val Ser 325 330 335 Ser Thr Met Lys Thr GlyLeu Leu Arg Ser Leu Phe Lys Asp Arg Phe 340 345 350 Cys Glu Glu Ser CysLys Glu Glu Thr Glu Ile Lys Phe His Ser Asp 355 360 365 Leu Leu Ser GlyIle Asn Ala Cys Tyr Asp Gly Asp Thr Val Ile Ile 370 375 380 Cys Pro GlyHis Tyr Val Val His Gly Thr Cys Ser Ile Ala Asp Ser 385 390 395 400 IleGlu Leu Glu Gly Tyr Gly Leu Pro Asp Asp Ile Val Ile Glu Lys 405 410 415Arg Gly Lys Gly Asp Thr Phe Val Asp Cys Thr Gly Met Asp Val Lys 420 425430 Ile Ser Gly Ile Lys Phe Ile Gln His Asp Ser Val Glu Gly Ile Leu 435440 445 Ile Ile His His Gly Lys Thr Thr Leu Glu Asn Cys Val Leu Gln Cys450 455 460 Glu Thr Thr Gly Val Thr Val Arg Thr Ser Ala Glu Leu Phe MetLys 465 470 475 480 Asn Ser Asp Val Tyr Gly Ala Lys Gly Ala Gly Ile GluIle Tyr Pro 485 490 495 Gly Ser Lys Cys Thr Leu Thr Asp Asn Gly Ile HisHis Cys Lys Glu 500 505 510 Gly Ile Leu Ile Lys Asp Phe Leu Asp Glu HisTyr Asp Ile Pro Lys 515 520 525 Ile Ser Met Ile Asn Asn Val Ile His AsnAsn Glu Gly Tyr Gly Val 530 535 540 Val Leu Val Lys Pro Thr Ile Phe CysAsp Leu Gln Glu Asn Thr Gln 545 550 555 560 Asp Glu Ile Asn Asp Asn MetVal Gln Lys Asn Lys Glu Ala Asp Val 565 570 575 Thr Glu Gly Leu Asp LeuGlu Glu Met Leu Gln Cys Val Ala Ser Lys 580 585 590 Met Glu Pro Tyr AlaThr Ala Asp Phe Asn Glu Gln Ala Lys Gly Asn 595 600 605 Cys Glu Ile IleAsn Glu Leu Leu Ala Ile Ser Met Gln Lys Gly Arg 610 615 620 Met Lys LysArg Leu Ser Glu Leu Gly Ile Thr Gln Ala Asp Asp Asn 625 630 635 640 IleMet Ser Gln Glu Met Phe Ile Glu Ile Met Gly Asn Gln Phe Lys 645 650 655Trp Asn Gly Lys Gly Ser Phe Gly Thr Phe Leu Tyr 660 665 14 amino acidsamino acid Not Relevant linear peptide 5 Met Val Pro Pro Arg Pro Asp LeuAla Ala Glu Lys Glu Pro 1 5 10 6 amino acids amino acid Not Relevantlinear peptide 6 Phe Leu Val Arg Glu Ser 1 5 24 base pairs nucleic acidsingle linear other nucleic acid /desc = “outer primer” 7 CCTCAGGGACCTTGCAGTCA GCTA 24 24 base pairs nucleic acid single linear othernucleic acid /desc = “nested primer” 8 CTTTCTCCAG AGACGCCAGC TCCT 24 24base pairs nucleic acid single linear other nucleic acid /desc = “outerprimer” 9 GACTACGCTG GAAAACTGTG TGCT 24 24 base pairs nucleic acidsingle linear other nucleic acid /desc = “nested primer” 10 ATGGGATCCATCACTGCAAG GAAG 24 24 base pairs nucleic acid single linear othernucleic acid /desc = “5′ mutagenic primer” 11 GAGTTCTTGG TGGCAGAGAG CACG24 24 base pairs nucleic acid single linear other nucleic acid /desc =“3′ mutagenic primer” 12 CGTGCTCTCT GCCACCAAGA AGTC 24

What is claimed is:
 1. A purified and isolated polypeptide encoded by apolynucleotides selected from the group consisting of: (a) thepolynucleotide of SEQ ID NO:1; (b) the polynucleotide of SEQ ID NO: 3;and (c) polynucleotides that hybridize under the high stringencyconditions of 650° C. in 3×SSC, 20 mM NaPO₄, and pH 6.8 followed bywashing at 65° C. in 0.015 M NaCl, 0.005 M sodium citrate, and 0.1% SDSto the complement (non-coding strand) of the polynucleotides of SEQ IDNO: 1 or SEQ ID NO: 3, wherein said polypeptide has the ability tostimulate cell growth or division in vitro.
 2. The polypeptide of claim1, wherein said polypeptide does not possess an amino terminalmethionine.
 3. The polypeptide of claim 1, further comprising one ormore chemical moieties attached thereto, optionally in a carrier.
 4. Thepolypeptide of claim 3, wherein said one or more chemical moieties is awater soluble polymer.
 5. The polypeptide of claim 4, wherein saidpolymer is selected from the group consisting of polyethylene glycol(PEG), monomethoxy-polyethylene glycol, propylene glycol homopolymers,polypropylene oxide/ethylene oxide copolymer, polyethylated polyols,polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6,-trioxane,ethylene/maleic anhydride copolymers, homopolymers of polyamino acids,random copolymers of polyamino acids, poly(n-vinylpyrrolidone)-polyethylene glycol, and polyvinyl alcohol.
 6. A purifiedand isolated polypeptide comprising SEQ ID NO:2, and biologically activefragments thereof wherein biologically activity is defined as having theability to stimulate cell growth or division in vitro.
 7. Thepolypeptide of claim 6, wherein said polypeptide does not possess anamino terminal methionine.
 8. The polypeptide of claim 6, furthercomprising one or more chemical moieties attached thereto, optionally ina carrier.
 9. The polypeptide of claim 8, wherein said one or morechemical moieties is a water soluble polymer.
 10. The polypeptide ofclaim 9, wherein said polymer is selected from the group consisting ofpolyethylene glycol (PEG), monomethoxy-polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide copolymer,polyethylated polyols, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6,-trioxane, ethylene/maleic anhydride copolymers, homopolymersof polyamino acids, random copolymers of polyamino acids, poly(n-vinylpyrrolidone)-polyethylene glycol, and polyvinyl alcohol.
 11. A purifiedand isolated polypeptide comprising SEQ ID NO:4, and biologically activefragments thereof wherein biologically activity is defined as having theability to stimulate cell growth or division in vitro.
 12. Thepolypeptide of claim 11, wherein said polypeptide does not possess anamino terminal methionine.
 13. The polypeptide of claim 11, furthercomprising one or more chemical moieties attached thereto, optionally ina carrier.
 14. The polypeptide of claim 13, wherein said one or morechemical moieties is a water soluble polymer.
 15. The polypeptide ofclaim 14, wherein said polymer is selected from the group consisting ofpolyethylene glycol (PEG), monomethoxy-polyethylene glycol, propyleneglycol homopolymers, polypropylene oxide/ethylene oxide copolymer,polyethylated polyols, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6,-trioxane, ethylene/maleic anhydride copolymers, homopolymersof polyamino acids, random copolymers of polyamino acids, poly(n-vinylpyrrolidone)-polyethylene glycol, and polyvinyl alcohol.
 16. A purifiedand isolated polypeptide comprising SEQ ID NO:2.
 17. A purified andisolated polypeptide comprising SEQ ID NO:4.